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NUV and Blue ps Diode Lasers

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1. High Performance Photon Counting User Manual NUV and Blue ps Diode Lasers Designed and manufactured in cooperation with Becker amp Hickl GmbH BDL SMC Picosecond Diode Lasers 1 p Becker amp Hickl GmbH Tel 49 30 787 56 32 FAX 449 30 787 57 34 y High Performance http www becker hickl com Photon Counting email info Ebecker hickl com BDL 375 SMC BDL 405 SMC BDL 440 SMC BDL 473 SMC NUV and Blue Picosecond Diode Lasers Picosecond pulsed operation or CW operation Free beam operation or coupling into optical fibre Correction of beam profile and astigmatism High power density in focused spot 60 of power delivered into single mode fibre Compatible with all commonly used fibre couplers Repetition rate from 20 MHz to 80 MHz Wavelengths of 375 nm 405 nm 440 nm and 473 nm Fast on off control multiplexing capability Excellent timing stability All driving and control electronics integrated Simple 12V power supply Compatible with the bh TCSPC systems AC e Designed an manufactured in cooperation with 14242472 2 BDL SMC Picosecond Diode Lasers Becker amp Hickl GmbH Nahmitzer Damm 30 12277 Berlin Germany Tel 49 30 787 56 32 FAX 49 30 787 57 34 http www becker hickl com email info O becker hickl com ACME bY ey LASOS Lasertechnik GmbH Carl Zeiss Promenade 10 07745 Jena Germany Tel 49 3641 2944 0 Fax 49 3641 29
2. Fig 35 Non photochemical quenching of chlorophyll in a leaf excited at 405 nm Fluorescence decay curves in different wavelength channels versus time of exposure 2 s per curve sequence starts from the back Extracted from same measurement data as Fig 34 BDL SMC Picosecond Diode Lasers 27 The non photochemical transients shown above occur on a time scale of several 10 seconds Good results are therefore obtained by recording a single sequence of decay curves at an acquisition time of a few seconds per curve The photochemical quenching transients are much faster Recording these transients requires a resolution of less than 100 us per step of the sequence Of course the number of photons detected in a time this short is too small to build up a reasonable decay curve Photochemical quenching transients must therefore be recorded by triggered sequential recording 8 9 The principle is shown in Fig 36 The excitation laser is periodically switched on an off via the laser off signal Each on phase initiates a photochemical quenching transient in the leaf each off phase lets the leaf recover Within each on phase a fast sequence of decay curves is recorded in the TCSPC module The measurement is continued for a large number of such on off cycles and the results are accumulated Experiment trigger to SPC module Recording Recording sequence sequence Leaf recovers Fig 36 Triggered sequenti
3. For low driving power the generator pulse initiates a damped sine wave voltage across the diode junction When the first positive peak reaches the forward conducting voltage of the diode current starts to flow through the junction As long as the laser threshold is not reached the light pulse is weak and broader than the current pulse If the driving power is increased the first positive peak drives a substantial forward current through the diode junction The dynamic impedance of the junction drops dramatically preventing the voltage at the junction to increase much above the forward voltage The current through the junction exceeds the laser threshold for a short fraction of the sine wave period and a short light pulse is emitted If the driving power is increased further the forward current pulse and consequently the light pulse becomes stronger The decrease in the dynamic resistance of the pn junction and the nonlinearity of the laser emission cause the optical pulse width to decrease Eventually the subsequent peaks of the sine wave Start to drive a forward current through the diode junction resulting in a tail or afterpulses of the light pulse Laser Vg Generator i h Rg L Diode Vj Generator Voltage medium g NY v Ci J Vg J Voltage accross I Laser Diode Junction Vg Generator voltage Rg Generator resistance L Lead inductance of diode Cj Junction capacitance of diode Seat through Laser Diode Junction No Light Emissio
4. Fluorescence Correlation Spectroscopy 30 Free beam operation 7 Jitter of pulse period 11 BDL SMC Picosecond Diode Lasers Key switch 6 Laser scanning microscope 29 Laser switch box 5 6 Multi mode fibres 8 Multiplexing lasers 16 BDL and BHLP lasers 17 by DDG 200 card 17 chlorophyll measurement 28 TCSPC system 17 On Off signal 4 27 28 Peak power 21 PMC 100 detector 24 29 Power average 21 control from DCC 100 card 14 influence on pulse shape 22 manual adjust 6 peak 21 22 pulse shape 22 software control 6 14 Power supply 4 5 ps mode 4 ps operation of laser diodes 20 Pulse period jitter 11 Pulse shape 20 22 Reference signal for TCSPC 11 Remote interlock connector 6 Reversed start stop 11 Safety emission indicators 6 key switch 6 labels 18 laser class 18 remote interlock 6 18 Shutdown 4 Single mode fibres 8 Specification BDL 375 SMC 33 BDL 405 SMC 34 BDL 440 SMC 35 BDL 473 SMC 36 connector pin assignment 37 dimensions 38 Stability of laser intensity 31 Status LEDs 5 Stop signal of TCSPC delay of stop pulses 12 stop pulse from correct pulse period 12 Switch box 5 6 control inputs 13 Synchronisation of TCSPC modules 11 Synchronisation signal 6 Synchronisation signal for TCSPC 11 TCSPC delay of stop signal 12 FLIM 29 fluorescence decay setup 24 BDL SMC Picosecond Diode Lasers multiplexing lasers 17 28 multi wavelength 25 26 29 reference signal 11 stop signal 11 synchronisation with laser 11 TCS
5. Moreover do not look into the laser beam through lenses binoculars microscopes camera finders telescopes or other optical elements that may collimate the light into your eye When using the lasers in combination with a microscope make sure that the beam path to the eyepieces is blocked for the laser wavelength when the laser is on If an optical fibre connected to a 3B laser has to be replaced the laser has to be turned off It is required to have a remote interlock connector that can be pulled to turn off the laser reliably In that case use the 15 pin connector at the laser side of the laser switch box The connector can be pulled off or plugged in at any time without causing damage to the laser BDL SMC Picosecond Diode Lasers 19 Fig 23 Remote interlock connector Pull the 15 pin connector at the laser side of the switch box to turn off the laser 20 BDL SMC Picosecond Diode Lasers Understanding Picosecond Diode Lasers Picosecond Operation of Laser Diodes The BDL SMC lasers are based on commercially available blue laser diodes 17 Picosecond pulsing of laser diodes requires to drive extremely short current pulses trough the pn junction of the diode Unfortunately commercial laser diodes are not optimised for this kind of operation In particular the junction capacitance C and the lead inductance L form an LC low pass filter that impedes a fast voltage rise across the diode junction The situation is shown in Fig 24
6. System Components The BDL SMC laser is shown in Fig 1 The power supply module is shown left It is a simple wall mounted 12V stabiliser Fig 1 middle shows the laser switch box The box contains the key switch mandatory for class 3B lasers and a switch to select between three pulse frequencies and CW operation Input connectors for control signals are located at the back of the switch box Via the control connectors the laser can switched on and off at us rate see Fig 15 page 15 Moreover the laser power can be controlled by an analog signal of 0 to 10V and the frequency can be switched by external TTL signals see Control Inputs page 13 Fig 1 BDL SMC laser Left Wall mounted power supply Middle Switch box with safety key switch for frequency and CW pulsed operation and control signal inputs Right Laser module containing the complete driving and control electronics Fig 1 right shows the laser module It contains the complete pulse generator and driver electronics the control electronics and an the active temperature stabilisation of the laser diode The diode itself is mounted on a peltier cooler inside the laser module The beam profile corrector is attached to the outside of the laser housing The front end of the corrector has threaded holes that fit to the standard 1 pitch of the commonly used fibre couplers or manipulators Fig 1 shows the BDL SMC laser with a fibre manipulator from Point Source UK
7. or open and the other pins are at low or connected to ground Important The frequency select pins are connected in parallel to the frequency select switch They can only be used when the frequency select switch is in the CW position In all other positions of the switch the pin corresponding to the frequency selected 1s connected to ground Please make sure that the source of the control signals connected to pin 2 3 and 4 is short circuit proof Pin 5 Ground Reference pin for all signals and power supply pin Pin 7 Laser Off Connecting this pin to TTL CMOS Low or GND switches the laser off The laser beam is shut down and the trigger output becomes inactive After disconnecting the pin from GND or switching to TTL CMOS high the laser resumes normal operation see Fig 15 Leave the pin open if you want the laser to run continuously Please notice that the laser does not deliver trigger pulses when it is switched off by Laser Off low For bh TCSPC modules this is no problem However if the Laser Off signal is pulsed at high rate the SPC module will display an average SYNC rate 1 e a rate lower than the frequency selected by the frequency selection switch The Laser OFF signal can also be connected to an SMA connector see Fig 13 The SMA input is connected in parallel with pin 7 of the sub D connector 14 BDL SMC Picosecond Diode Lasers Pin 12 Power Bias An input voltage applied to pin 1
8. 27 through Fig 30 show pulse shapes pulse width and peak power for different BDL SMC lasers It should be noted here that the Nichia laser diodes used in the BDL lasers are continuously improved For example the optical output power of the 405 nm diodes has been increased by a factor of 10 within two years At the same time there was a substantial improvement in efficiency i e in the output power for a given forward current Fig 27 through Fig 30 should therefore considered to demonstrate the general relation between power and pulse parameters not the quantitative values Pulse shapes for a BDL 405 SMC laser for different average optical power at 50 MHz are shown in Fig 27 50 MHz 0 4 mW 50 MHz 0 8 mW 50 MHz 1 2 mW 50 MHz 1 6 mW FWHM 127 ps FWHM 84 ps FWHM 62 ps FWHM 32 ps Fig 27 Pulse shapes for a BDL 405 SMC laser at 50 MHz Recorded with Hamamatsu R3809U 50 MCP 12 and BH SPC 730 TCSPC module 9 The curves were recorded with a Hamamatsu R3809U 52 MCP PMT and a bh SPC 730 TCSPC module The R3809U 52 was operated at 3 kV yielding an instrument response function IRF of 30 ps fwhm Important The instrument response function of the R3809U 52 has a shoulder of about 300 ps duration 8 9 12 The afterpulse visible in the recorded pulse shape in a large part comes from that shoulder not from the laser pulse itself The pulse width decreases continuously with increased output power The best pulse shape for 80 MH
9. 488 nm beam path Unfortunately replacing the 488 nm Argon laser with the 473 nm diode laser is often not acceptable Therefore please contact bh or the manufacturer of your microscope before you consider attaching a 473 nm laser to it Wiring diagrams for a single detector FLIM system and a multi spectral FLIM system are shown in Fig 40 left and right For details please see 6 and 9 Beam Blanking Beam Blanking from Microscope from Microscope Power Supply Laser Power Contro z ER Laser Power Control ower Supply 4 o ar S va o O s 0 Con 2 wer Power Supply 8 Control lt Con 3 Single Mode Fibre into Microscope Single Mode Fibre into Microscope Laser Sync Output PML SPEC Assembly Laser Sync Output Fibre Filter Adapter Scan Clock PMC _from Fibre d 100 Microscope from H Scan Fibre fro Scan Head Head Power Supply amp Control Scan Clocks from Microscope Fig 40 Wiring diagrams of a single detector FLIM system left and a multi spectral FLIM system right For FLIM the excitation power required in the focus of the microscope objective lens is not particularly high Many samples can be exited with less than 50 uW However in scanning microscopes the back aperture of the objective is often over illuminated to obtain a uniform intensity distribution over the whole aperture Moreover a substantial fraction of the excitation light gets lost in the
10. Beam diameter before coupler Polarisation Coupling efficiency into fibre typically Stability of Repetition Rate Pulse to Pulse Jitter Reaction time to Laser on signal pulsed mode Reaction time to Laser on signal CW mode Power and pulse shape stabilisation after switch on Fibre coupler Trigger Output Pulse Amplitude Pulse Width Output Impedance Connector Delay from Trigger to Optical Pulse Jitter between Trigger and Optical Pulse Control Inputs Frequency 20 MHz Frequency 50 MHz Frequency 80 MHz CW operation Laser ON Off External Power Control Power Supply Power Supply Voltage Power Supply Current Power Adapter Mechanical Data Dimensions Mounting Thread Maximum Values Power Supply Voltage Voltage at Digital Control Inputs Voltage at Ext Bias Input Ambient Temperature 1 Typical values sample tested Depends on pulse width and selected power 20 50 80 MHz or CW operation 370 nm to 380 nm typ 375 nm 50 to 90 ps 10 to 100 mW 20 MHz 0 05 mW to 0 16 mW 50 MHz 0 1 mW to 0 4 mW 80 MHz 0 15 mW to 0 6 mW CW mode 0 5 mW to 5 mW 0 7 mm TEMop mode horizontal 20 100 ppm lt 20 ps l us 3 us 3 min all 1 footprint couplers Point Source Scha fter amp Kirchhoff OZ Optics Linus 100 to 300 mV peak into 50 Q 1 ns 50 Q SMA lt 500 ps lt 10 ps TTL CMOS high gt TTL CMOS high gt TTL CMOS high gt TTL CMOS high g
11. Excitation Wavelength Multiplexing page 27 18 BDL SMC Picosecond Diode Lasers Laser Safety The BDL SMC lasers are class 3B laser products The laser safety regulations dictate that the lasers be labelled with the stickers shown in Fig 20 and that the labels and the location of the labels on the lasers be described in the manual The laser class is indicated on the laser by an explanatory label Fig 20 left The laser aperture is marked with the aperture labels Fig 20 middle and right Fig 20 Left to right Explanatory label aperture labels Moreover each laser has a manufacturer identification as shown in Fig 21 A Becker amp Hickl GmbH Nahmitzer Damm 30 12277 Berlin Germany www becker hickl com BDL 405 SMC Picosecond Diode Laser 405 nm CW 20 50 80 MHz S N 95 1015 Manufactured December 2005 Complies with FDA performance standards for laser products except for deviations pursuant to Laser Notice No 50 dated July 26 2001 Fig 21 Manufacturer identification label The position of the labels on the laser modules is shown Fig 22 Fig 22 Location of the labels on the lasers Laser safety regulations forbid the user to open the housing of the laser or to do any maintenance or service operations at or inside the laser Use of controls or adjustments or performance of procedures other than specified herein may result in hazardous radiation exposure or damage to the laser module
12. Status Indicators Connectors and Controls The back panel of the laser head is shown in Fig 2 The left LED indicates that the laser is active The LED flashes when the power of the laser is on and the Laser Off signal is high or unconnected The other two LEDs show the status of the cooler of the laser diode The right LED is on when the cooling of the laser diode is active It may turn off after some time of operation when the diode has been cooled down and almost no cooling power is required to hold it at constant temperature The red LED in the middle turns on when the cooling power is high It normally turns off after some minutes of operation The 15 pin sub D connector connects the power supply and control signals from the laser switch box to the laser The lasers are delivered with appropriate connecting cables so that user access to the 15 pin connector is not normally needed For pin assignment please see page 37 6 BDL SMC Picosecond Diode Lasers Power amp Control Laser Cooling TRG Out On High Active Power Bias od A at 9 x A ee LAJ r ci is a f t Fig 2 Back panel of the BDL SMC laser E t A trigger output signal is available at an SMA connector The shape of the signal is shown in Fig 10 page 11 Depending on the laser power he amplitude of the synchronisation signal may vary between about 100 and 300 mV There are two potentiometers at the back
13. Voltage Power Supply Current Power Adapter Mechanical Data Dimensions Mounting Thread Maximum Values Power Supply Voltage Voltage at Digital Control Inputs Voltage at Ext Bias Input Ambient Temperature 1 Typical values sample tested Depends on pulse width and selected power 20 50 80 MHz or CW operation 436 nm to 448 nm typ 440 nm 40 to 90 ps 40 to 250 mW 20 MHz 0 07 mW to 0 2 mW 50 MHz 0 3 mW to 1 mW 80 MHz 0 4 mW to 1 2 mW CW mode 1 mW to 20 mW 0 7 mm TEMop mode horizontal 60 100 ppm lt 20 ps l us 3 us 3 min all 1 footprint couplers Point Source Sch fter8Kirchhoff OZ Optics Linus 5 100 to 300 mV peak into 50 Q 1 ns 50 Q SMA lt 500 ps lt 10 ps TTL CMOS high gt TTL CMOS high gt TTL CMOS high gt TTL CMOS high TTL CMOS low gt analog input 0 to 10V 9 V to 12 V 300 mA tol A AC DC power adapter with key switch and control box in cable 160 mm x 90 mm x 60 mm two M6 holes 0 V to 15 V 2 V to 7 V 12 V to 12 V 0 C to 40 C gt 35 2 Recommended power adjust range Lower power gives broader pulses higher power gives ringing in pulse shape Power levels above the given range can be selected but may impair the lifetime of the laser diode 3 All inputs have 10 KQ pull up resistors Open input is equivalent to logic high 4 Dependent on ambient temperature Cooling current changes due to temperatur
14. bit of the digital outputs Connector 2 is used to switch the laser on and off Laser OFF Signal The BDL SMC lasers can be switched on and off by applying a TTL CMOS signal to pin 7 of the sub D connector TTL Low or connecting the pin to GND switches the laser off The Laser OFF signal works both in the picosecond mode and in the CW mode The reaction times are typically ps mode CW mode Laser OFF low to emission on lt lus 3 us Laser OFF high to emission off lt 100 ns 3 us The on off behaviour is shown in Fig 15 The curves were recorded by a pin photodiode connected to a Tektronix TDS 3052 oscilloscope Important If you want to test the switching behaviour apply a reverse bias to the photodiode and switch the oscilloscope to an input impedance of 50 Q To obtain sufficient signal amplitude at 50 Q it may be necessary to use an avalanche photodiode BDL SMC Picosecond Diode Lasers 15 nVOQwM4 00Ns A Ch 0mY10 M4 00u4s A Ch 2482 100mY QAM 4 0045 A Ch U 22 40 i U 22 40 Ii U 22 40 Fig 15 On Off behaviour of the BDL SMC laser Left to right CW mode 20 MHz 80 MHz Upper trace laser intensity lower trace Laser OFF signal Recorded with avalanche photodiode connected to Tektronix TDS 3052 Time scale 4 us per division The variation of the shape of the laser pulses after the off on transition is shown in Fig 16 40 us TTL high pulses were applied to the Laser OFF input and the sequence was accumu
15. delay is shown in Fig 12 Fluorescence Detector transit time Time interval to be recorded Reference pulse Photon pulses from Detector from laser undelayed Delay to be inserted in reference channel Previous Delayed TAC stop reference pulse reference delayed pulse Time Fig 12 Reversed start stop should be used with a delay in the reference channel to stop the TAC with the correct laser pulse 9 Typical signal transit times of detectors are MCP PMTs 1 ns Hamamatsu R7400 TO 8 PMTs 5 6ns Hamamatsu H5783 photosensor modules 5 6 ns bh PMC 100 detector module 5 6ns 30 mm side window PMTs 25 ns XP 2020 linear focused PMTs 25 ns A good stop delay to start with is 15 ns or 3 m cable for an MCP PMT and 25 ns or 5 m cable for TO 8 PMTs Please see 9 for details BDL SMC Picosecond Diode Lasers 13 Control Inputs The control input connectors at the switch box are shown in Fig 13 Power Laser OFF External Control Connector Fig 13 Control inputs The pin assignment of the external control connector is l not connected 9 not connected 2 Frequency 20 MHz 10 not connected 3 Frequency 50 MHz 11 not connected 4 Frequency 80 MHz 12 Power Bias 0 to 10 V 5 GND 13 not connected 6 not connected 14 not connected 7 Laser Off 15 GND 8 not connected Pin 2 3 4 Frequency select pins Frequency select pins CMOS compatible The laser works at the selected frequency when the corresponding pin is at high
16. effects can further be reduced by tilting the laser or placing a polariser in the beam path Unfortunately the exact angles depend on the numerical aperture of the detection light path which is not exactly predictable The PMC 100 detector is controlled by a DCC 100 detector controller The DCC 100 provides the power supply for the PMT module the preamplifier and the cooler of the PMC 100 Moreover it provides software controlled detector gain and overload shutdown The photon pulses of the PMC 100 are connected directly to the TCSPC module Any bh SPC module can be used The stop timing reference signal comes from the trigger output of the laser An example of a fluorescence decay measurement is shown in Fig 32 Stilben blue curve and Rhodamin 110 red curve were excited by a BDL 405 SMC laser The black curve is the IRF obtained from a scattering solution BDL SMC Picosecond Diode Lasers 25 Fig 32 Fluorescence decay curves of stilben blue and rhodamin 110 red excited at 405 nm black Time scale 3 ns per division time channel width 12 ps Despite of its simplicity the setup features high sensitivity and good time resolution Another advantage is that light reflected at the emission filter and the photocathode of the detector is not focused back into the sample Therefore the setup has less problems with optical reflections then more complex optical systems In all fluorescence measurement that use deconvolution o
17. in 1972 15 Theory and applications of FCS are described in 22 The required femtoliter volume can be obtained by one photon excitation and confocal detection or by two photon excitation see Fig 42 The principle is the same as in a laser scanning microscope A continuous or high repetition rate laser beam is focused into the sample through the microscope objective lens The fluorescence light from the sample is collected by the same lens separated from the laser by a dichroic mirror and fed through a pinhole in the upper image plane of the microscope lens In a confocal microscope the fluorescence light from above or below the focal plane is not focused into the pinhole and therefore substantially suppressed With a high aperture objective lens the effective sample volume is of the order of a femtoliter with a depth of about 1 5 um and a width of about 400 nm Due to its good beam quality the BDL SMC lasers are excellently suitable for FCS experiments The laser can be either free beam coupled into the microscope or fibre coupling may be used FCS experiments and other single molecule techniques especially benefit from the capability of the BDL SMC lasers to be operated both in the ps and in the CW mode Thus combined FCS lifetime experiments 10 or burst integrated fluorescence lifetime BIFL experiments 21 can be performed in the ps mode whereas pure correlation experiments can take advantage of the high power available in the CW mo
18. scanner and the microscope optics The high coupling efficiency of the BDL SMC lasers is therefore a considerable benefit There is sufficient power margin and the laser can be operated at a power that yields optimum pulse shape Fig 41 shows fluorescence lifetime images of plant tissue recorded in a Zeiss LSM 510 upgraded with a bh BDL 405 SMC laser and SPC 830 TCSPC module Plant tissue makes good test samples because it contains a wide variety of different fluorophores The fluorescence decay functions are 30 BDL SMC Picosecond Diode Lasers therefore multi exponential Left to right Fig 41 shows images of the lifetime of the fast component the lifetime of the slow component and the ratio of the amplitudes of the fast and the slow component of the fluorescence Fig 41 Fluorescence lifetime images of plant tissue Double exponential decay analysis left to right Lifetime of fast component lifetime of slow component ratio of amplitudes of fast and slow component bh BDL 405 SMC laser Zeiss LSM 510 bh SPC 830 TCSPC module Fluorescence Correlation Spectroscopy FCS Fluorescence correlation spectroscopy FCS is based on exciting a small number of molecules in a femtoliter volume and correlating the fluctuations of the fluorescence intensity The fluctuations are caused by diffusion rotation intersystem crossing conformational changes or other random effects The technique dates back to a work of Magde Elson and Webb published
19. the fluctuation of the number of molecules in the focus Fig 44 shows autocorrelation curves of the laser intensity for a BDL 405 SMC laser The curves resemble FCS results obtained from a samples that does not show any intrinsic intensity fluctuations Fluctuations at times shorter than 1 ms would show up as bumps in the curves fluctuations at longer time scales as an offset from C 1 No such effects are visible in Fig 44 That means the BDL SMC lasers can be used for FCS down to correlation coefficients smaller than 1 001 32 BDL SMC Picosecond Diode Lasers AE FCS Display a aj ibi sPc 830 FCS Display 15 1 010 1 010 1 008 1 008 1 006 1 006 1 004 1 004 5 1 002 5 1 002 3 s z a k a 5 1 000 mn 3 1 000 CIN EC A A II t D E S 0 998 0 998 0 996 0 996 0 994 0 994 0 992 0 992 0 990 1 1 1 1 0 990 1 1 1 i 1 0 1 1 0 10 0 100 0 1000 0 0 1 1 0 10 0 100 0 1000 0 10000 0 Time ps Time ps Fig 44 Autocorrelation FCS curves of the laser intensity BDL 405 SMC laser recorded over 5 minutes with R3809U MCP PMT and SPC 830 TCSPC module Left pulsed operation Right CW operation Scale of correlation coefficient from 0 99 to 1 01 BDL SMC Picosecond Diode Lasers Specification BDL 375 SMC Optical Repetition Rate Wavelength Pulse Width FWHM at 1 mW power 50 MHz Peak Power Average Power Average CW equivalent power user adjustable
20. 2 changes the bias of the laser diode The voltage can be in the range of 0 V to 10 V The output power increases with the voltage The Power Bias signal can also be connected to an SMA connector see Fig 13 The SMA input is connected in parallel with pin 12 of the sub D connector Pin 15 Ground Reference pin for all signals and power supply pin Software Control of the Laser Power The Power Bias input can be used for electronic power control of the BDL SMC lasers The power control signal is connected either to an SMA connector or to pin 12 of a 15 pin connector both located at the back of the laser switch box Controlling the laser power electronically is particularly convenient in TCSPC systems that use the bh DCC 100 detector controller 3 see Fig 14 left Connector 1 Connector2 Connector 3 guy suy muv Mon Laser sv ml sv MHo Sai Detector 2 j TE f bt er Es les gt ats ews 3 _ 5V E SV E s zw ii eho on off Mi power supply qn LAS ES AS ne E 3 ash 2 Ce Sx 55 oF ot oF os i r 3 os AS 3 NMXD0512S0 e o a o OVLD r gt 00 no pl cet g Laser power 0 EDO 62 25 ain HV Fig 14 Left DCC 100 detector controller card Right DCC 100 software panel Fig 14 right shows the software control panel of the DCC 100 The Connector 1 channel is used to control the power of the laser while Connector 3 controls the detector The b7
21. 44 17 info lasos com www lasos com Ist Edition May 2006 This manual is subject to copyright However reproduction of small portions of the material in scientific papers or other non commercial publications is considered fair use under the copyright law It is requested that a complete citation be included in the publication If you require confirmation please feel free to contact Becker amp Hickl BDL SMC Picosecond Diode Lasers 3 Contents ONCE Wind 4 General DESCAPUO eps oca caoio stes 5 Sy Stemi ODDONE IIS ie a R a a sli 5 Status lidicators Connectors and Emol a o ewes 5 VASTOS WAUCI BOX a A A A A A A A ENTO EANA 6 Operating the DI SIMIC East 7 Eres B e amO Pera DN A se aenee tua eeaee autores 7 PD COOPIN ss ad se a a N copiS 8 Als mnent ot Pont Source COUPE espida E E E N tuts 8 A onment of OZ Optics COMPITE A asco 10 IP Atraces te centered a aiuaaetiuainaad dated esldousunde sceade de a a Rutoneaatdeeseantlancaseuntees 11 COnTOlINpUS i a E Mada 13 Sotware Gonmonor the Laser POW eK uoin T A cala 14 aser CO Sn E E e E r E a TE A EEAS 14 TESPC Systems with the BDL SME Tasers ice a a a aa a a N a 16 IVI Gp he Sn Laser iia 16 Laser Salcido oie 18 Understandine Picosecond Diode Last cds 20 Picosecond Operation Of Laser DiC eG kia cect a ticas 20 Average Power atid Peak POWER ida 21 Pulse SHAPE A act EA AAA ci 22 Application to Fluorescence Lifetime SpectrosCOpy cccccccccccnonononononncnnnnonnnonononnnonnnnnnnnn
22. C Picosecond Diode Lasers flavinoids Due to the Kautski effect the fluorescence intensity and lifetimes vary with the time of exposure and are thus different for different excitation wavelength and different excitation intensity It is therefore difficult to obtain comparable results for different excitation conditions An experiment that avoids this problem is shown in Fig 38 The sample is excited by two lasers A BDL 405 SMC and a BHLP 700 650 nm are multiplexed The light from the sample is split into a 515 nm and a 700 nm component by a dichroic mirror and two bandpass filters The fluorescence components are detected by two PMT modules The PMTs are connected to the TCSPC module via a HRT 41 router 1 Thus both fluorescence components are recorded simultaneously One routing bit is required to separate the photons of both detectors A second routing bit is used to separate the photons excited by the two lasers The stop signal for the TCSPC module comes from the synchronisation outputs of the lasers Because only one laser is active at a time the pulses can be combined by a simple power combiner Multiplexin l i i Laser 1 Synchronisation Power combiner 405 nm from lasers BDL 405 SMC Dichroic Bandpass mirror filter 700 nm Multiplexing E a E SPC 830 TCSPC module stop Detector 1 a PMC 100 mE routing wt Detector 2 PMC 100 BHLP 700 Fig 38 Simultaneous measurement at two excitation and two emission wavelen
23. Coupling efficiency into fibre typically 60 Stability of Repetition Rate 100 ppm Pulse to Pulse Jitter lt 20 ps Reaction time to Laser on signal pulsed mode l us Reaction time to Laser on signal CW mode 3 us Power and pulse shape stabilisation after switch on 3 min Fibre coupler all 1 footprint couplers Point Source Scha fter amp Kirchhoff OZ Optics Linus Trigger Output Pulse Amplitude 100 to 300 mV peak into 50 Q Pulse Width 1 ns Output Impedance 50 Q Connector SMA Delay from Trigger to Optical Pulse lt 500 ps Jitter between Trigger and Optical Pulse lt 10 ps Control Inputs Frequency 20 MHz TTL CMOS high gt Frequency 50 MHz TTL CMOS high gt Frequency 80 MHz TTL CMOS high gt CW operation TTL CMOS high Laser ON Off TTL CMOS low External Power Control analog input 0 to 10V Power Supply Power Supply Voltage 9Vto 12 V Power Supply Current 300 mA to 1 A Power Adapter AC DC power adapter with key switch and control box in cable Mechanical Data Dimensions 160 mm x 90 mm x 60 mm Mounting Thread two M6 holes Maximum Values Power Supply Voltage 0 V to 15 V Voltage at Digital Control Inputs 2 V to 7 V Voltage at Ext Bias Input 12 V to 12 V Ambient Temperature 0 C to 40 C 1 Typical values sample tested Depends on pulse width and selected power 2 Recommended power adjust range Lower power gives broader pulses higher power gives ri
24. H Fie Nad Oe a Pulse Inverter crnstennatannstannstnanssnsnatnanstnanstncnntansnsnnsshannsbensnbennhenannennhensthenashansshannansnashsnashennshenasinanstnansinenshasnatnanstnnnssnsnstncestnassharsshenannensnhensnbensshennthensnnennshsnsshsnsshensshannsnssnshnenshvenshnenatven A Fig 10 Left and middle Electrical pulse from the trigger output 50 mV div left 10 ns div middle 2 ns div Recorded with Tektronix TDS 3052 Oscilloscope Right A PPI pulse inverter The polarity of the trigger pulse is positive To connect the pulse into the SYNC input of a bh TCSPC module please use an A PPI pulse inverter see Fig 10 right The adapter is delivered with all bh TCSPC modules The time difference between the trigger pulse and the light pulse is less than 1 ns and does not change appreciably for different output power and for different repetition rate The shift of the light pulse with the power referred to the trigger pulse is shown in Fig 11 The repetition rate was 50 MHz the power was varied between 0 3 mW and 1 3 mW The total shift with the power is about 200 ps Fig 11 Shift of the light pulse with the output power referred to the trigger pulse Left to right Power 0 3 mW 0 5 mW 0 8 mW and 1 3 mW Recorded with bh SPC 730 TCSPC module 9 and Hamamatsu R3809U MCP PMT 12 When using the BDL SM lasers in conjunction with the bh TCSPC modules please keep in mind that the TCSPC modules use reversed start stop R
25. H Routing modules for time correlated single photon counting manual available on www becker hickl com Becker amp Hickl GmbH PML 16 C 16 channel detector head for time correlated single photon counting user handbook available on www becker hickl com 2006 Becker amp Hickl GmbH DCC 100 detector control module manual available on www becker hickl com Becker amp Hickl GmbH MSA 200 MSA 300 MSA 1000 Photon counters multiscalers application manual www becker hickl com 2002 Becker amp Hickl GmbH BHL 600 and BHLP 700 Red and Near Infrared Picosecond Diode Laser Modules available on www becker hickl com Becker amp Hickl GmbH Modular FLIM Systems for Zeiss LSM 510 Laser Scanning Microscopes 2005 available on www becker hickl com W Becker A Bergmann M A Hink K Konig K Benndorf C Biskup Fluorescence lifetime imaging by time correlated single photon counting Micr Res Techn 63 58 66 2004 W Becker Advanced time correlated single photon counting techniques Springer Berlin Heidelberg New York 2005 W Becker The bh TCSPC handbook Becker amp Hickl GmbH 2005 available on www becker hickl com W Becker A Bergmann E Haustein Z Petrasek P Schwille C Biskup L Kelbauskas K Benndorf N Kl cker T Anhut I Riemann K K nig Fluorescence lifetime images and correlation spectra obtained by multi dimensional TCSPC Micr Res Tech 69 186 195 2006 S Felekyan R Kiihnemuth
26. L SMC Picosecond Diode Lasers Connector Pin Assignment all BDL Lasers External control connector at the laser switch box 1 not connected 9 2 Frequency 20 MHz 10 3 Frequency 50 MHz 1 4 Frequency 80 MHz 12 5 GND 13 6 not connected 14 7 Laser Off 15 8 not connected 37 not connected not connected not connected Power Bias 0 to 10 V not connected not connected GND 15 pin connectors at the laser and at the left side of the laser switch box 1 not connected 9 2 Frequency 20 MHz 10 3 Frequency 50 MHz 1 4 Frequency 80 MHz 12 5 GND 13 6 not connected 14 7 Laser Off 15 8 Test output internal 7V 9 pin connector at the right side of the laser switch box l 12 V from power supply 6 2 12 V from power supply 7 3 not connected 8 4 GND 9 5 GND Test output internal bias 12 V from power supply not connected Power Bias 0 to 10 V not connected not connected GND 12 V from power supply not connected GND GND 38 BDL SMC Picosecond Diode Lasers Dimensions Fig 45 BDL SMC lasers dimensions in mm Laser shown with Point Source Coupler Mounting plane of fibre coupler Beam output 25 4 88 Fibre coupler mounting plane Fig 46 Left Mounting plane of fibre coupler Right Bottom view with M6 mounting holes BDL SMC Picosecond Diode Lasers 39 References 10 11 12 13 14 15 16 17 18 19 20 ZA De 23 Becker amp Hickl Gmb
27. PC system with BDL SMC 16 wiring diagram 16 29 Trigger output 6 11 41
28. V Kudryavtsev C Sandhagen W Becker C A M Seidel Full correlation from picoseconds to seconds by time resolved and time correlated single photon detection Rev Sci Instrum 76 083104 2005 Hamamatsu Photonics K K R3809U 50 series Microchannel plate photomultiplier tube MCP PMTs 2001 R Kiihnemuth C A M Seidel Principles of single molecule multiparameter fluorescence spectroscopy Single Molecules 2 2001 251 254 A Liebert H Wabnitz D Grosenick R Macdonald Fiber dispersion in time domain measurements compromising the accuracy of determination of optical properties of strongly scattering media J Biomed Opt 8 512 516 2003 D Magde E Elson W W W Webb Thermodynamic fluctuations ina reacting system measurement by fluorescence correlation spectroscopy Phys Rev Lett 29 705 708 1972 K Maxwell G N Johnson Chlorophyll fluorescence a practical guide Journal of Experimental Botany 51 659 668 2000 S Nakamura S F Chichibu Introduction to nitride semiconductor blue lasers and light emitting diodes Taylor amp Francis 2000 D V O Connor D Phillips Time Correlated Single Photon Counting Academic Press London 1984 OZ Optics Ltd Operating instructions Laser to fibre coupler with adjustable focus 2002 Point Source Ltd Kineflex fibre manipulator operating instructions M Prummer B Sick A Renn U P Wild Multiparameter microscopy and spectroscopy for single molecule analysis A
29. al recording of photochemical quenching transients The laser is cycled on and off Each on phase starts a photochemical quenching transient in the leaf A sequence of waveform recordings is taken within each on phase A large number of such on off cycles is accumulated to obtain enough photons with the individual steps of the accumulated sequence A typical result is shown in Fig 37 The on time was 2 5 ms Within this time a sequence of 50 decay curves of 50 us collection time each was recorded The off time was 90 ms 20 000 of such on off cycles were accumulated Fig 37 Photochemical quenching of chlorophyll in a leaf Fluorescence decay curves in different wavelength channels versus time Triggered sequential recording 50 us per curve 20 000 measurement cycles accumulated Excitation Wavelength Multiplexing Excitation wavelength multiplexing is used to excite different fluorophores during the same measurement Compared with sequential measurement at different excitation wavelengths fast multiplexing has the advantage that changes in the fluorescence behaviour of the sample have the same effect on all fluorescence signals recorded A typical application of multiplexed excitation are measurements at living plants Green leaves show the typical chlorophyll fluorescence around 700 nm and a blue green fluorescence from 28 BDL SM
30. ape is obtained by using high driving amplitudes and a bias as low negative as possible It should be noted that the operating conditions of picosecond pulsed laser diodes are different from those of modulated laser diodes used in communication equipment A modulated laser diode is always forward biased and there is a continuous forward current through the laser diode Consequently the diode junction has a low dynamic impedance that shorts the junction capacitance The speed of the diode is then determined mainly by the lead inductance and the generator impedance Average Power and Peak Power The typical pulse width for a picosecond laser diode is in the range of 40 to 100 ps For a repetition rate in the 20 to 80 MHz range the duty factor is on the order of 300 As shown in Fig 26 the result is a relatively high peak power even for low average CW equivalent power Pp peak power Pa Fig 26 Relation between peak power average power pulse width and pulse period 22 BDL SMC Picosecond Diode Lasers For ps diode lasers the optical peak power is far beyond the permissible steady state power for the laser diodes used Due to the short pulse width the high peak power does not cause any thermal damage However damage may also occur by extremely fast nonlinear optical effects It is therefore recommended to avoid unnecessarily high peak power and not to exceed the power at which substantial afterpulses develop Pulse Shape Fig
31. de 11 FCS and BIFL experiments can be performed efficiently in the FIFO or Time Tag mode of the bh TCSPC modules Please see 8 9 for details An example of a combined FCS lifetime measurement is shown in Fig 43 BDL SMC Picosecond Diode Lasers 31 Pinhole Dichroic Mirror Detector Tube Lens Objective Lens Sample Excited Detected Fig 42 Fluorescence correlation spectroscopy Left Basic optical setup Right Beam waist of laser and confocal detection volume 23 22 As Il i 21 a os A n 818 E A g17 A 16 O 2 TA ka 7 EE A 7 O ON j A S NI ee I 11 a 1 0 ama 1 I 1 1 10 100 1000 10000 Time ps J Fig 43 Combined FCS Lifetime measurement of a dye solution Left Fluorescence decay curve Right FCS curve Once the optical system is setup correctly FCS measurements on highly diluted dye solutions on the order of 10 mol l are relatively easy This is not necessarily the case for FCS measurement in living cells Especially in transfected cells the fluorophore concentration cannot be accurately controlled It is usually much higher than required for FCS The number of molecules in the focus can easily be on the order of 100 or even 1000 resulting in an extremely small amplitude of the correlation function 10 Reasonable FCS results from such specimens can of course only be obtained if the fluctuation of the laser power are smaller than
32. e regulation of laser diode 5 Operation below 13 C may result in extended warm up time Caution Class 3B laser product Avoid direct eye exposure Light emitted by the device may be harmful to the human eye Please obey laser safety rules when operating the devices Complies with US federal laser product performance standards 36 BDL 473 SMC BDL SMC Picosecond Diode Lasers Optical Repetition Rate 20 50 80 MHz or CW operation Wavelength 467 nm to 476 nm typ 473 nm Pulse Width FWHM at 1 mW power 50 MHz 40 to 90 ps Peak Power 40 to 250 mW Average Power 20 MHz 0 07 mW to 0 2 mW Average CW equivalent power 50 MHz 0 3 mW to 1 mW user adjustable 80 MHz 0 4 mW to 1 2 mW CW mode 0 5 mW to 10 mW Beam diameter before coupler 0 7 mm TEM oo mode Polarisation horizontal Coupling efficiency into single mode fibre typically 60 Stability of Repetition Rate 100 ppm Pulse to Pulse Jitter lt 20 ps Reaction time to Laser on signal pulsed mode l us Reaction time to Laser on signal CW mode 3 us Power and pulse shape stabilisation after switch on 3 min gt Fibre coupler all 1 footprint couplers Point Source Sch fter8Kirchhoff OZ Optics Linus Trigger Output Pulse Amplitude 100 to 300 mV peak into 50 Q Pulse Width 1 ns Output Impedance 50 Q Connector SMA Delay from Trigger to Optical Pulse lt 500 ps Jitter between Trigger and Optical Pulse lt 10 ps Contr
33. er A Stop Excitation to TCSPC A SPC 630 730 830 BDL SMC Laser Emission PMC 100 Power supply filter PMT module and control Fig 31 Fluorescence lifetime measurement setup For samples with strong scattering possible background emission of the laser diode may be removed by a bandpass filter in the excitation path Moreover 1t may be convenient to have variable ND filter placed in the excitation path After passing the filters the laser beam is sent into the sample cell The fluorescence light is detected at an angle of 90 from the excitation beam The detector is a bh PMC 100 module It is controlled via a DCC 100 detector controller card The PMC 100 is located close to the sample cell The fluorescence light is collected directly 1 e without an additional transfer lens The detection wavelength interval is selected by a bandpass filter at the input of the detector Important In the BDL SMC lasers the polarisation of the laser beam is horizontal Sending a horizontally polarised laser beam into the sample and detecting the fluorescence under an angle of 90 from the excitation would result in detecting only I components of the fluorescence 1 e projections of the electrical field vectors perpendicular to the polarisation of the laser This would result in large distortion of the measured decay functions by rotational depolarisation 8 9 18 In the setup shown in Fig 31 the laser is therefore turned by 90 Polarisation
34. eriments by fluorescence correlation anti bunching and burst integrated fluorescence lifetime or multi parameter spectroscopy 11 13 21 By switching between CW and ps operation these techniques can be applied to the same sample or in some cases even to the same molecules All BDL SMC lasers come with a beam corrector that corrects both for beam shape and astigmatism The lasers are compatible with all commonly used single mode fibre couplers Due to the excellent correction of the beam up to 70 of the laser power can be coupled into a single mode fibre Thus high laser power can be focused into a diffraction limited spot The lasers are thus excellent excitation sources for single molecule spectroscopy and time resolved laser scanning microscopy The BDL laser modules have a TTL controlled shutdown input that can be used to switch the laser off and on within a time of lus Thus lasers of different wavelength can be multiplexed at microsecond periods In laser scanning microscopes the shutdown function is used to switch off the laser during the line and frame flyback The BDL SMC lasers are operated from a simple wall mounted 12 V power supply The complete control and driving electronics is contained in the laser module The design of the BDL SMC lasers results in exceptionally low RF noise radiation and low timing drift between the electrical trigger output and the light pulses BDL SMC Picosecond Diode Lasers 5 General Description
35. eversed start stop operation of TCSPC requires a reference pulse at the end of the signal period or better at the end of the recorded time interval 12 BDL SMC Picosecond Diode Lasers 8 9 At the high repetition rate of the BDL SMC lasers the next laser pulse is no more than 50 ns away so that a reasonable recording is achieved without problems However it is not always clear which laser pulse actually stops the time measurement It can happen that the stop pulse is not the same laser pulse that excited the detected photon but a pulse from a period before or after Stopping with a pulse from a different period is no problem if the laser pulses have a constant period and no pulse to pulse jitter The BDL SMC lasers have however selectable pulse periods Moreover the clock oscillator of a diode laser may have a pulse to pulse jitter of some 10 ps If the reference pulses come from the wrong signal period the position of the recorded signal in the TAC range changes when the laser period is changed Moreover the pulse to pulse jitter adds to the transit time spread of the TCSPC system To stop the TAC with the correct laser pulse the reference signal should be delayed so that the reference pulse arrives after a photon pulse from the same period 8 9 The correct delay in the reference channel is the detector transit time plus the width of the recorded time interval plus a few ns for the TAC start delay The relation of the detector and reference
36. f the fluorescence data from the instrument response function IRF the laser must be operated at the same power for the fluorescence measurement and the IRF measurement As shown in Fig 27 page 22 to Fig 30 the shape of the laser pulse changes with the power Changing the laser power between the recordings may therefore lead to a wrong shape of the IRF and consequently to a poor fit of the data and large lifetime errors for fast lifetime components Autofluorescence of Tissue A simple optical setup for single point multi spectral measurements of tissue autofluorescence is shown in Fig 33 left A BDL 405 SMC or a BDL 375 SMC laser is used for excitation A fibre probe is used to excite the sample and to collect the fluorescence light The probe contains 7 multi mode fibres of 0 5 mm diameter The central fibre delivers the laser the surrounding fibres collect the fluorescence The detection system consists of a bh PML Spec multi wavelength assembly and an SPC 830 TCSPC module The detection system records decay curves in 16 wavelength intervals simultaneously Multi spectral fluorescence decay data of human skin obtained this way are shown in Fig 33 right PML SPEC detetcor assembly ps Diode Polychromator Laser Single Ue lan TCSPC Module Fibre Filter 420nm LP with Routing Fibre bundle BDL 405 SMC 4 YA Grating section W Becker amp Hickl of LOT MS125 SPC 830 bundle 600 nm Fig 33 Left Optica
37. g Microscopy Laser scanning microscopes can relatively easily be upgraded for fluorescence lifetime imaging by multi dimensional TCSPC 8 9 FLIM is especially easy with multiphoton microscopes 7 10 The Ti Sapphire laser of these microscopes is an almost ideal excitation source for FLIM Standard confocal microscopes however use only continuous lasers Upgrading a standard confocal microscope with FLIM therefore requires a suitable pulse excitation source to be added 23 All confocal microscopes couple the visible lasers into the optical beam path via single mode fibres Most of the microscopes use the coupler and fibre manipulator systems of Point Source Ltd UK Attaching a BDL SMC laser to a confocal microscope is therefore relatively easy The only requirement is that a free input fibre be available Many microscopes have a 405 nm continuous diode laser integrated The excitation problem of FLIM 1s then easily solved by attaching the fibre of this laser to a BDL 405 SMC Experiments requiring high continuous power can in most cases by performed in the CW mode of the BDL 405 SMC It is therefore not necessary to switch the fibre between the BDL 405 SMC and the diode laser of the microscope Lasers of 440 or 473 nm wavelength can in principle used in the same way However coupling the laser light into the sample requires a dichroic beamsplitter of the correct wavelength in the scanner optics The 473 nm laser can often be coupled via the
38. g signal inputs of the TCSPC module are used to direct the photons of the individual lasers into separate photon distributions To simplify the generation of the multiplexing and routing signals the DDG 200 digital delay generator card is available see Fig 18 The card is controlled via the software panel shown in Fig 18 right BDL SMC Picosecond Diode Lasers 17 P Main Parameters Start Stop Exitl ME RepeatSequence E Endiess Delayed Pulses PEE Channel s Polari m m be i a Repeat Time 10ns No of cycles Wie a ol O E 2000 3 1 View Edit Chan Data a a a Bll Start Pulse Width 10ns Settings from ddg_auto sel f J 00 O SevusvevevvsssesesvevevsssvuseseusvsssveveveeussssseueveGeVESESUSDOVEUTSSSESUDEVESEFESUSUSUEUUNTSSEUDOUUSUFUSESEUOUUUUSSSEDUSUUNOSFNSSESEEUEUUSSSTUUSUUEOFESEDUESOUUSUUSUSUUNSUENETUSHTUSEEUSNESEEYS Fig 18 Left DDG 200 card for multiplexing control Right Software panel of the DDG 200 The DDG 200 can be programmed to multiplex up to four lasers Multiplexing periods can be programmed at any time scale from a few 100 ns to 10 milliseconds The control signals can be defined non overlapping 1 e with gaps of some 100ns duration between the individual lasers This guarantees that any crosstalk is avoided even if the routing and multiplexing signals get delayed in the connecting cables A TCSPC system with two multiplexed lasers is shown schematically in Fig 19 A bh DDG 200 card is used
39. gths A typical result is shown in Fig 39 Fluorescence decay curves for a fresh leaf are shown left results for a dry leaf right The synchronisation signal of the 650 nm laser was delayed by 3 ns to make the curves better distinguishable The multiplexing period was 50 ms At this rate the lifetime is modulated by photochemical quenching but not by non photochemical quenching Therefore different lifetimes of the 695 nm emission are obtained for both wavelengths No such effect is seen in the 695 nm emission of the dry leaf The green emission at 515 nm from the fresh leaf has a considerably lower intensity a shorter lifetime and a multi exponential decay profile This indicates that a strong non uniform quenching process 1s at work Both the intensity and the lifetime of the green emission increase in the dry leaf 10000 650 gt 695 nm 650 gt 695 nm 405 gt 695 nm 405 gt 695 nm 405 gt 515 nm 1000 M hyi Ya 10 1 1 i 1 1 1 1 1 t 1 1 1 0 0000 1 5000 3 0000 4 5000 6 0000 7 5000 9 0000 10 5000 12 0000 13 5000 15 0000 Time ns 10 0 0000 1 5000 3 0000 4 5000 6 0000 7 5000 9 0000 10 5000 12 0000 13 5000 15 0000 Time ns Fig 39 Dual wavelength excitation and dual wavelength detection of the fluorescence of a fresh leaf left and a dry leaf right Multiplexed excitation at 405 nm and 650 nm dual detector recording at 515 nm and 695nm BDL SMC Picosecond Diode Lasers 29 Laser Scannin
40. hing between 20 50 and 80 MHz and CW operation Please note that the frequency switch must be in the CW position when electronic frequency control is used For pin assignment and signal specification please see Control Inputs page 13 The 15 pin connector at the laser side can be used as a remote interlock connector The connector can be pulled off or plugged in at any time without causing damage to the laser BDL SMC Picosecond Diode Lasers 7 Operating the BDL SMC Lasers Free Beam Operation The BDL SMC lasers can be used both in free beam or fibre coupled systems For free beam operation the lasers are used without a fibre coupler attached see Fig 4 Fig 4 Free beam operation of the BDL SMC laser The beam diameter is about 0 7 mm The beam profile at 1 m distance from a BDL 405 SM laser is shown in Fig 5 The definition of the profile improves with increasing power Because of the high peak power in the pulsed mode the beam profile is slightly better than in the CW mode Section X Section Y Be ey 7 Fig 5 Beam profile in 1m distance from the laser BDL 405 SMC CW mode 30 mW The collimator and beam correction optics of the BDL SMC lasers is aligned during manufacturing Once the optics are aligned the elements are fixed in place permanently Therefore please do not attempt to change anything in the optics of the laser If you need focusing other beam diameters or other beam shape plea
41. l setup for single point autofluorescence measurement Right Multi spectral fluorescence decay data of human skin Time scale O to 15 ns wavelength scale 410 to 600 nm intensity scale logarithmic from 500 to 30 000 counts per channel 26 BDL SMC Picosecond Diode Lasers The count rates obtained from biological tissue are surprisingly high At an excitation wavelength of 405 nm a count rate of 2 10 s could be obtained at an excitation power of only 60 uW Recording Chlorophyll Transients The fast on off switching capability of the BDL SMC lasers can be used to record excitation driven transient fluorescence phenomena Typical examples of transient fluorescence effects are the Kautski effect or the fluorescence transients of chlorophyll in living plants 16 When a dark adapted leaf is exposed to light the intensity of the chlorophyll fluorescence starts to increase After a steep rise the intensity falls again and finally reaches the steady state level The rise time is of the order of a few milliseconds to a second the fall time can be from several seconds to minutes The initial rise of the fluorescence intensity is attributed to the progressive closing of reaction centres in the photosynthesis pathway Therefore the quenching of the fluorescence by the photosynthesis decreases with the time of illumination with a corresponding increase of the fluorescence intensity The fluorescence quenching by the photosynthesis pathway is ter
42. lated in the Scan Sync Out mode of an SPC 830 TCSPC module for 10 on off transitions 9 The photons were detected by an R3809U 52 MCP PMT 12 Each curve of the sequence represents an interval of 500 ns As can be seen from Fig 16 right the pulse shape is stable after 2 us The shift of the pulses within the first 2 us is less than 30 ps Scan Pixel X Fig 16 Transient pulse shape variation of the BDL SMC lasers after transition from Laser OFF low to Laser OFF high 40 us TTL high pulses were applied to the Laser OFF input Repetition rate 50 MHz SPC 830 TCSPC module with R3809U MCP PMT triggered sequential recording in Scan Sync In mode Each curve of the sequence represents an interval of 500 ns Left Curve plot Right Contour plot 16 BDL SMC Picosecond Diode Lasers TCSPC Systems with the BDL SMC Lasers A wiring diagram of a TCSPC system with a BDL SMC laser a PMC 100 detector a DCC 100 detector controller and an SPC TCSPC module is shown in Fig 17 PMC Power supply Detector amp ovid cable Laser SPC module BDL SMC cm ae power supply Fig 17 Connection diagram of a TCSPC system with a BDL SMC laser a PMC 100 detector a DCC 100 detector controller and an SPC module The DCC 100 module controls both the laser and the detector The laser power can be changed via a control signal from connector 1 of the DCC 100 The emission is switched on and off via co
43. med photochemical quenching The slow decrease of the fluorescence intensity at later times is termed non photochemical quenching Results of a non photochemical quenching measurement are shown in Fig 34 The fluorescence in a leaf was excited by a bh BDL 405 SMC picosecond diode laser The fluorescence was detected by a bh PML SPEC multi wavelength detection assembly 2 see Fig 33 Decay curves in the 16 wavelength intervals of the PML SPEC were recorded by a bh SPC 803 TCSPC module Simultaneously with the switch on of the laser a recording sequence was started in the TCSPC module 30 recordings were taken in intervals of 2 seconds Fig 34 shows four selected steps of this sequence The decrease of the fluorescence lifetime with the time of exposure is clearly visible Fig 34 Non photochemical quenching of chlorophyll in a leaf excited at 405 nm Recorded wavelength range from 620 to 820 nm time axis 0 to 8 ns logarithmic display normalised on peak intensity Left to right O s 20 s 40 s and 60 s after start of exposure Fig 35 shows fluorescence decay curves at selected wavelengths versus the time of exposure extracted from the same measurement data set as Fig 34 The sequence starts at the back and extends over 60 seconds Also here the decrease of the fluorescence lifetime with the time of exposure is clearly visible
44. n KL in S N Fig 24 Junction voltage Vj and junction current Ij in a picosecond laser diode for different driving pulse amplitude Vg The behaviour of the junction current explains why there is a relation between the pulse shape and the pulse power Good pulse shapes can be obtained only at moderate optical power Using a stronger laser diode does not generally help It can actually make the situation worse because the junction capacitance of the larger laser diode is higher BDL SMC Picosecond Diode Lasers 21 An additional control parameter is obtained by adding a bias voltage to the driving pulse For blue laser diodes which have a forward conducting voltage of 4 to 5 V the bias can be positive in forward direction or negative in reverse direction The influence of the diode bias is shown in Fig 25 Generator eee Vg R L Diode y Generator Voltage Vi Voltage accross Bias 3 Laser Diode Bias 2 Junction Bias 1 Vg Generator voltage lj Current through Laser Diode Rg Generator resistance Junction Ly Lead inductance of diode Cj Junction capacitance of diode V bias Bias voltage Light Emission Fig 25 Junction voltage Vj and junction current Ij in a picosecond laser diode for different diode bias voltage Positive bias results in higher output power but makes afterpulsing more likely Reverse bias helps to suppress afterpulses but reduces the power In general the best optical pulse sh
45. nal Chem 76 1633 1640 2004 R Rigler E S Elson eds Fluorescence Correlation Spectroscopy Springer Verlag Berlin Heidelberg New York 2001 A Riick F Dolp C Happ R Steiner M Beil Fluorescence lifetime imaging FLIM using ps pulsed diode lasers in laser scanning microscopy Proc SPIE 4962 44 2003 40 Index Alignment of fibre coupler OZ Optics 10 Point Source 9 A PPI pulse inverter 11 Autofluorescence of tissue 25 Average power 21 Beam diameter 7 Beam profile 7 Beam profile corrector 4 5 7 Bias adjust 6 Chlorophyll transients 26 27 Confocal detection 30 Connectors for control signals 6 pin assignment 37 trigger output 6 Control inputs 6 connector pin assignment 13 37 external power control 14 frequency select 13 laser off signal 13 14 on off reaction times 14 CW mode 4 DCC 100 card 14 24 29 DDG 200 card 16 Delay of stop signal of TCSPC 12 Delayed start stop operation of TCSPC 12 Dimensions 38 Emission indicators 6 FCS 30 confocal detection 30 fluctuation of laser intensity 31 focal volume 30 optical system 30 pinhole 30 Fibre fibre manipulator 5 Fibre coupler 8 alignment OZ Optics 10 alignment point source 9 OZ Optics 10 Point Source 8 FLIM 29 excitation power 29 single mode fibre coupler 29 wiring diagram of system 29 Fluorescence autofluorescence of tissue 25 depolarisation 24 lifetime experiments 24 lifetime imaging 29 multi spectral detection 25 26 of chlorophyll 26 27
46. nging in pulse shape Power levels above the given range can be selected but may impair the lifetime of the laser diode 3 All inputs have 10 KQ pull up resistors Open input is equivalent to logic high 4 Dependent on ambient temperature Cooling current changes due to temperature regulation of laser diode 5 Operation below 13 C may result in extended warm up time Caution Class 3B laser product Avoid direct eye exposure Light emitted by the device may be harmful to the human eye Please obey laser safety rules when operating the devices Complies with US federal laser product performance standards BDL SMC Picosecond Diode Lasers BDL 440 SMC Optical Repetition Rate Wavelength Pulse Width FWHM at 1 mW power 50 MHz Peak Power Average Power Average CW equivalent power user adjustable Beam diameter before coupler Polarisation Coupling efficiency into single mode fibre typically Stability of Repetition Rate Pulse to Pulse Jitter Reaction time to Laser on signal pulsed mode Reaction time to Laser on signal CW mode Power and pulse shape stabilisation after switch on Fibre coupler Trigger Output Pulse Amplitude Pulse Width Output Impedance Connector Delay from Trigger to Optical Pulse Jitter between Trigger and Optical Pulse Control Inputs Frequency 20 MHz Frequency 50 MHz Frequency 80 MHz CW operation Laser ON Off External Power Control Power Supply Power Supply
47. nnector 1 see also Fig 14 The trigger output pulses of the lasers are inverted by an A PPI pulse inverter and fed into the SYNC stop input of the SPC module A cable of 5 m length is used to place the stop pulse after the photon pulses originating from photons detected in the signal period of a particular trigger pulse see Fig 12 A PMC 100 detector is used to detect the light from a sample The PMC 100 is controlled via connector 3 of the DCC 100 The DCC 100 controls the gain of the detector provides the power supply for the internal preamplifier high voltage generator and cooler of the PMC 100 In case of overload the DCC 100 shuts down the high voltage of the PMT of the PMC 100 The single photon pulses of the PMC 100 are fed into the CFD start input of the SPC module A large number of modifications of the TCSPC setup are possible The detector may be replaced with an ultra fast R3809U MCP PMT with a single photon avalanche photodiode or with a multi spectral detector assembly For recording fluorescence lifetime images the TCSPC system may also be connected to a laser scanning microscope see Laser Scanning Microscopy page 29 For details please see 9 Multiplexing Lasers By controlling several lasers via their Laser OFF inputs the lasers can be multiplexed at periods down to the microsecond range Normally laser multiplexing is used in conjunction with multiplexed TCSPC operation 8 9 That means the routin
48. nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnan 24 Pluorescence liten me Ex PEE ia 24 AUTO UOrescence OL dis ic 25 Recording Chlorophyll Transients sandia da ado 26 Excitation Wavelenota Wilt plexin 9 id A ads 2 Laser Scanmine Microscopy aisre a a a a a a SA 29 Fluorescence Correlation Spectroscopy PES id 30 SPECIAL isles A A AR AA A AA acatanest 33 BOE AI A o A BO OSO DECESO RI O E E Reese O EAA 33 BOLAS ME inci 34 BDE MOSME aiii 35 BDLAT IS MG turmalina ad oia 36 Connector Pin Assiznmentall BOL Lasers 00 E a E 37 Dita ne nados 38 RETON OS aaa E O autos ellis ia ates ae ae a sons 39 4 BDL SMC Picosecond Diode Lasers Overview The BDL 375 SMC BDL 405 SMC BDL 440 SMC and BDL 473 SMC lasers emit at typical wavelengths of 375 nm 405 nm 440 nm and 473 nm respectively The lasers can be switched between picosecond pulse operation and CW operation In the picosecond mode pulse repetition rates of 20 MHz 50 MHz and 80 MHz can be selected The pulse width is in the range of 40 ps to 90 ps the CW equivalent power between 0 2 and 1 5 mW The high repetition rate and the short pulse width make the BDL SMC lasers ideally suited for time correlated single photon counting TCSPC applications 8 9 In the CW mode an output power of up to 40 mW is available The lasers can thus be used for a number of applications that require both continuous excitation at high power and excitation with ps pulses Typical examples are single molecule exp
49. of the laser module The Power adjust changes the operating voltage of the driving generator The Bias adjust changes the bias voltage of the laser diode Higher voltage and higher positive bias give higher output power The best pulse shape is obtained with minimum negative bias and high driving power Please see also Picosecond Operation of Laser Diodes page 20 Please note that the power of the BDL SMC lasers can also be changed by an analog input signal By using the DCC 100 detector controller card of the bh TCSPC systems 3 9 the laser power can be controlled per software see Fig 14 page 14 Controlling the power per software is not only more convenient than turning a potentiometer but also makes it easier to restore previously used settings Laser Switch Box The laser switch box is shown in Fig 3 The box contains the mandatory laser safety elements of class 3B lasers a key switch and emission indicators Laser action is indicated by four LEDs of different coulour at least one of them being visible through any laser safety eyewear The Laser Off LED shows the status of the laser on off signal Fig 3 Laser switch and connection box The connectors for the control signals are shown in Fig 3 right There are two SMA connectors one for the on off signal and one for the analog power control signal The same signals can be connected to a 15 pin sub D connector This connector has also inputs for switc
50. ol Inputs Frequency 20 MHz TTL CMOS high gt Frequency 50 MHz TTL CMOS high gt Frequency 80 MHz TTL CMOS high gt CW operation TTL CMOS high Laser ON Off TTL CMOS low External Power Control analog input 0 to 10V Power Supply Power Supply Voltage 9Vto 12 V Power Supply Current 300 mA to 1 A Power Adapter AC DC power adapter with key switch and control box in cable Mechanical Data Dimensions 160 mm x 90 mm x 60 mm Mounting Thread two M6 holes Maximum Values Power Supply Voltage 0 V to 15 V Voltage at Digital Control Inputs 2 V to 7 V Voltage at Ext Bias Input 12 V to 12 V Ambient Temperature 0 C to 40 C 1 Typical values sample tested Depends on pulse width and selected power 2 Recommended power adjust range Lower power gives broader pulses higher power gives ringing in pulse shape Power levels above the given range can be selected but may impair the lifetime of the laser diode 3 All inputs have 10 KQ pull up resistors Open input is equivalent to logic high 4 Dependent on ambient temperature Cooling current changes due to temperature regulation of laser diode 5 Operation below 13 C may result in extended warm up time Caution Class 3B laser product Avoid direct eye exposure Light emitted by the device may be harmful to the human eye Please obey laser safety rules when operating the devices Complies with US federal laser product performance standards BD
51. re To align the fibre coupler proceed as follows 20 1 2 3 4 5 6 Insert the alignment tool as indicated in Fig 8 and adjust Al and B1 for maximum throughput Reverse the alignment tool and adjust A2 and B2 for maximum throughput Repeat step 1 After step 3 the optical axis of the fibre manipulator is aligned with the axis of the laser beam Insert the fibre Adjust Al and B1 for maximum output intensity Adjust Al and A2 for maximum intensity This step is a lateral shift of the optical axis Therefore turn both screws in the same direction until you find the setting that yields maximum intensity Adjust Bl and B2 for maximum intensity This step is a lateral shift of the optical axis Therefore turn both screws in the same direction until you find the setting that yields maximum intensity 10 BDL SMC Picosecond Diode Lasers Alignment of OZ Optics Coupler The OZ Optics coupler consists of two plates separated by a resilient O ring The plate holding the fibre receptacle is tilted against the laser head adapter by three screws 1 2 3 see Fig 9 left The laser head adapter contains a lens that focuses the laser beam into the core of the fibre By adjusting the three screws one can move the core of the fibre laterally with respect to the focus 19 To adjust the focus longitudinally the connector of the fibre has a differential thread see Fig 9 right When the adjustment is complete the front plate is locked again
52. rews 1 2 3 until you obtain maximum intensity and a symmetrical intensity pattern on the paper 9 Tighten the tilt screws evenly while maintaining maximum intensity and a symmetrical intensity pattern No more than 1 27 mm 0 05 space should be left between the base adapter and the coupler flange 10 Remove the multi mode fibre and put in the single mode fibre At least a small amount of light should be transmitted through the fibre Adjust the tilt screws 1 2 3 until you obtain maximum throughput Important The Airy disk pattern in the focus of the lens may have secondary maxima If the throughput is unacceptably low make sure that you are in the central peak of the Airy disk 11 Adjust the focus by rotating the focus adjustment of the fibre connector It may be necessary to readjust the tilt screws BDL SMC Picosecond Diode Lasers 11 12 Once the best focus is achieved tighten the nut that locks the focus adjustment 13 Turn in the three lock screws a b c until the just make contact with the laser head adapter 14 Tighten the lock screws by an additional quarter turn If the throughput drops adjust the lock screws Slightly until optimum coupling efficiency is restored Trigger Output TCSPC measurements require a timing reference signal from the laser 8 9 To minimise timing drift the BDL SMC lasers derive a trigger signal directly from the laser diode The electrical pulse shape is shown in Fig 10 Hicki Gmb
53. s four adjustment screws Al A2 B1 and B2 Inside the manipulator the fibre input adapter is pressed against the alignment screws by a spring loaded counter bearing Thus the fibre adapter can both be shifted and tilted by turning the adjustment screws Under normal use e g after removing and re inserting the fibre only fine adjustments are required It is then sufficient to adjust the front screws A2 and B2 for maximum image intensity Do not turn the screws by more than 1 2 turn Once the manipulator is totally misaligned you have to go through the complete alignment procedure BDL SMC Picosecond Diode Lasers 9 Beam Profile Fibre Manipulator Corrector Alignment Screws B1 B2 Input Collimator of Fibre Ak Alignment Tool Alignment Screws Fig 7 Front end of the BDL 405SM laser Beam profile corrector fibre manipulator with alignment screws input adapter of the single mode fibre and alignment tool The complete alignment procedure is illustrated in Fig 8 For the first steps an alignment tool is required see Fig 7 The tool is a tube which has a pinhole in the optical axis insert alignment tool this side first Step 5 adjust A1 and A2 turn screws in same direction insert alignment tool this side first ll Step 3 Repeat step 1 Step 6 adjust B1 and B2 adjust A1 and B1 turn screws in same direction insert alignment tool this side first Fig 8 Steps of the alignment procedu
54. se use external optics The intensity distribution in the focus of a 200 mm lens is shown in Fig 6 gt Ar Section X Section Y bald EAEG Fig 6 Intensity distribution in the focus of a 200 mm lens BDL 405 SMC CW mode 30 mW 8 BDL SMC Picosecond Diode Lasers Fibre Coupling Optical fibres consist of a core with high index of refraction and a cladding with lower index of refraction The light is kept inside the core of the fibre by total internal reflection Optical fibres come in two different versions multi mode fibres and single mode fibres Multi mode fibres have diameters from typically 50 um to 1 mm Any light within a given input cone is transferred to the output Coupling light into a multi mode fibre is therefore relatively easy However rays of different angles to the optical axis have different optical path lengths and consequently different transit times Therefore a light pulse coupled into a multi mode fibre spreads out in time For a fibre of Im length illuminated at maximum numerical aperture the transit time spread is on the order of 100 ps The transit time spread is independent of the diameter of the fibre 14 The second drawback of multi mode fibres is that the light leaves the end of the fibre from the whole cross section of the core and within a cone of large angle Focusing the light from the fibre into a small spot at best yields a de magnified image of the core cross section A diffraction limi
55. st the base plate by tightening the lock screws a b c The longitudinal focus adjustment is locked by tightening the focus lock nut see Fig 9 right Focus lock Focus adjustment Fig 9 Left OZ Optics coupler with adjustment screws 1 2 3 and lock screws a b c Right Fibre connector with focus adjustment For fine adjustment it is normally sufficient to loosen the lock screw a b c by a quarter turn and turn the adjust screws 1 2 3 until maximum intensity is transmitted through the fibre Then tighten the lock screws one after another in small steps see below alignment step 14 If the coupler is totally misaligned so that no light is transmitted through the fibre proceed as described below 19 7 Without a fibre examine the output on a sheet of paper If the image on the screen is not centred adjust the lateral position of the focusing lens with respect to the laser beam To do this loosen the tilt adjustment screws 1 2 3 by about half a turn and apply lateral pressure to the side of the coupler flange This will shift the lateral position of the coupler flange with respect to the laser head adapter Once the image has been centred tighten the tilt adjustment screws to their original position 8 Insert a multi mode fibre of a core diameter of 50 to 100 um Observe the transmitted light on a sheet of paper Make sure that the lock screws a b c are not pressing against the base plate Adjust the tilt sc
56. t TTL CMOS low gt analog input 0 to 10V 9 V to 12 V 300 mA tol A AC DC power adapter with key switch and control box in cable 160 mm x 90 mm x 60 mm two M6 holes 0 V to 15 V 2 V to 7 V 12 V to 12 V 0 C to 40 C gt 33 2 Recommended power adjust range Lower power gives broader pulses higher power gives ringing in pulse shape Power levels above the given range can be selected but may impair the lifetime of the laser diode 3 All inputs have 10 KQ pull up resistors Open input is equivalent to logic high 4 Dependent on ambient temperature Cooling current changes due to temperature regulation of laser diode 5 Operation below 13 C may result in extended warm up time Caution Class 3B laser product Avoid direct eye exposure Light emitted by the device may be harmful to the human eye Please obey laser safety rules when operating the devices Complies with US federal laser product performance standards 34 BDL 405 SMC BDL SMC Picosecond Diode Lasers Optical Repetition Rate 20 50 80 MHz or CW operation Wavelength 401 nm to 410 nm typ 405 nm Pulse Width FWHM at 1 mW power 50 MHz 50 to 90 ps Peak Power 80 to 500 mW Average Power 20 MHz 0 12 mW to 0 6 mW Average CW equivalent power 50 MHz 0 3 mW to 1 6 mW user adjustable 80 MHz 0 4 mW to 2 4 mW CW mode 5 mW to 40 mW Beam diameter before coupler 0 7 mm TEM oo mode Polarisation horizontal
57. ted spot cannot be obtained Single mode fibres have core diameters on the order of 3 um Because of the small diameter only a single wave mode is transmitted through the fibre Coupling into a single mode fibre is difficult not only because of the small diameter but also because of the relatively small angle of the input light cone However the light leaves the fibre output as a single wave mode That means the light virtually comes from an infinitely small spot Light transmitted by a single mode fibre can therefore focused into a diffraction limited spot Moreover because only a single wave mode is transmitted there is virtually no transit time spread Because of the superior features of single mode fibres the BDL SMC lasers are available with single mode fibre coupling The lasers are compatible with almost any of the commonly used single mode fibre couplers Alignment of Point Source Coupler The fibre coupling system of Point Source Ltd UK uses special fibres that have a focusing lens permanently attached to the fibre input Both the lens and the fibre are assembled in a cylindrical adapter that is inserted into a fibre manipulator see Fig 7 The Point Source system thus avoids any alignment at the sub um scale 20 The result is high efficiency and extraordinarily good long term stability Due to the good long term stability commercial laser scanning microscope almost exclusively use the Point Source system The fibre manipulator ha
58. to generate the ON signals for the lasers and routing signals for the SPC module The control sequence is shown in the lower right corner The lasers are switched on alternatingly Simultaneously with the laser switching a bit at the routing input of the SPC card is toggled between 0 and 1 Consequently the SPC module records the signals of the two lasers into different memory blocks Laser calada dl Detector CS Al PMC Power PA DCC 100 O gt ovid cable PMC 100 HE combiner m I i ee on T 1 DDG 200 I i power supply Control sequence BDL SMC or BHLP Routing Trigger gg ON gt Laser 1 Laser ON power supply Laser 2 BDL SMC or BHLP Fig 19 TCSPC system with two multiplexed lasers Please note that multiplexing can be used even in combination with multidetector operation sequential recording and imaging by TCSPC scanning techniques 9 Moreover the bh blue BDL SMC lasers can be multiplexed with the BHLP 700 red and NIR lasers 5 An application to chlorophyll measurements is described in 8 A BDL 405 405 nm and a BHLP 700 650 nm laser are multiplexed and the signals at 540 nm and 700 nm are recorded simultaneously by two detectors The setup is able to record both the fluorescence of flavins and of chlorophyll independently of photochemical and non photochemical quenching transients see also
59. z 50 MHz and 20 MHz is obtained at 1 6 mW 1 mW and 0 4 mW respectively Typical curves of the peak power and the pulse width are shown in Fig 28 BDL SMC Picosecond Diode Lasers 23 0 0 2 0 4 0 6 0 8 1 0 1 2 1 4 average CW equivalent power Fig 28 Pulse width and peak power for a BDL 405 versus average power at 50 MHz repetition rate Pulse width corrected for 30ps IRF width of detection system Typical pulse shapes of the BDL 440 SMC and the BDL 473 SMC are shown in Fig 29 and Fig 30 BDL 440 SMC 50 MHz 0 3 mW FWHM 61 ps bh SPC 830 TCSPC module with Hamamatsu R3809U MCP PMT FWHM values corrected for IRF width of 30 ps Fig 29 Pulse shapes for a BDL 440 SMC laser at 50 MHz Recorded with Hamamatsu R3809U 50 MCP and bh SPC 830 TCSPC module 50 MHz 0 3 mW 0 5 mW FWHM 64 ps FWHM 41 ps FWHM 63 ps FWHM 36 ps bh SPC 830 TCSPC module with Hamamatsu R3809U MCP PMT FWHM values corrected for IRF width of 30 ps Fig 30 Pulse shapes for a BDL 473 SMC laser at 50 MHz Recorded with Hamamatsu R3809U 50 MCP and bh SPC 830 TCSPC module 24 BDL SMC Picosecond Diode Lasers Application to Fluorescence Lifetime Spectroscopy Fluorescence Lifetime Experiments The BDL SMC lasers in conjunction with the bh TCSPC modules make fluorescence lifetime measurements an easy task A simple fluorescence lifetime system is shown schematically in Fig 31 Trigger Pulse invert

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