Selective Plane Illumination Microscope

Fluorescence Imaging Laboratory, Kyiv, Ukraine

__L-SPIM v0__

L-SPIM v0 parameters overview

Parameter	Value
Detection objective	Olympus PlaN 10x 0.25
Magnification	5.56x (F100) or 6.94x (F125)
Camera	Basler Ace 2R Pro (a2A5320-23umPRO), 5320x3032 px (16.1 Mpx)
Pixel size	0.493 μm/px (F100) or 0.395 μm/px (F125)
Field of view	2.62x1.49 mm (F100) or 2.1x1.2 mm (F125)
Z-stack step resolution	16 μstep/μm (0.0625 μm/μstep)
Lateral resolution, FWHM theoretical/estimated	1.12 μm/2.06 μm
Axial resolution, FWHM theoretical/estimated	11.70 μm/ 6.32 μm
Excitation lasers	510 nm, 640 nm
Emission filters	Chroma HQ545/40m, Chroma D620/20m, Chroma HQ700/75m

Detection arm

Detection params

Parameter	Value
Detection objective	Olympus PlaN 10x 0.25 FN 22 (F18)
Detection tube lens	F100 or F125
Magnification	5.56x or 6.94x
Camera	Basler Ace 2R Pro (a2A5320-23umPRO)
Pixel size	0.493 μm/px or 0.395 μm/px
Field of view	2.62x1.49 mm or 2.1x1.2 mm
Theoretical resolution (500 nm)	1.220 μm
Theoretical depth of field (500 nm, n=1.33)	10.640 μm
Estimated resolution FWHM (F100)	2.06 μm
Estimated depth of field FWHM (F100)	6.32 μm
Z-stack step (Sutter Instruments MPC-200 with MP225/M)	0.0625 μm/μstep (16 μstep/μm)

Filters

Excitation arm

Light path

Illumination objective 4x (F45)

Full FOV light sheet parameters:

• Tubus lens F125 FOV high 2.1 mm => d = 2.1 => $NA_{ill}$ = d / 2f = 2.1/(2*45) = 0.023

$x_{ls} \approx \frac{2 n \lambda}{\pi NA_{ill}^2} = \frac{2 * 1.33 * 510}{3.14 * 0.023^2} = 817 \mu m$

$z_{ls} \approx \frac{2 \lambda}{\pi NA_{ill}} = \frac{2 * 510}{3.14 * 0.023} = 14.1 \mu m$

Lasers power

• 510 nm: 2.2 mW in imaging chamber
• 640 nm: 1.2 mW in imaging chamber

Control software

Micromanager configuration

Camera

Model	Basler Ace 2R
ID	a2A5320-23umPRO
Sensor	Sony IMX542 (CMOS)
Sensor size	14.58x8.31 mm (1.1")
Sensor diagonal	16.78 mm
Pixels (HxV)	5320x3032 (16.1 Mpx)
Pixel size	2.74x2.74 μm
Frame rate	up to 24 fps

GPIO pinout

Pin	Function	I/O Line	Colour
1	12–24 VDC camera power	-	Brown
2	Opto-coupled I/O input line	Line 1	White
3	Ground for opto-coupled I/O line	-	Blue
4	General purpose I/O (GPIO) line	Line 2	Black
5	General purpose I/O (GPIO) line	Line 3	Grey
6	Ground for camera power and General Purpose I/O (GPIO) lines	-	Pink

Basler Power-I/O Cable

GPIO configuration

Pylon Viewer v7.2 camera parameters, category Digital I/O Control:

• Line Selector: Line 2
• Line Inverter: not selected
• Line Mode: Output
• Line Source: Exposure Active

Sync

ArdiunoMM

• Bauderate: 57600
• Responce timeout: 2000 ms

Arduino32Bit (deprecated)

Synchronisation with Arduino32bitBoards, specs for ESP32:

• Baudrate: 115200
• Laser TTL trigger module requires 5V, need level conversion 3.3V -> 5V

Pinout:

• Trigger: Pin 5 - camera Line 2 input
• Channel 1 (index 0): Pin 25/DAC - Level Converter ON
• Channel 2 (index 1): Pin 26/DAC - 510 nm trigger
• Channel 3 (index 2): Pin 27/PWM - 640 nm trigger
• Channel 4 (index 3): Pin 15/PWM
• Channel 5 (index 4): Pin 14/PWM
• Channel 6 (index 5): Pin 4/PWM
• Channel 7 (index 6): Pin 23/PWM
• Channel 8 (index 7): Pin 19/PWM

Decimal switch state value for ON is 2^index.

Switch states

State	Pin Hight	State
Level Converter ON	25	1
510 nm	25+26 (Ch.1+Ch.2)	1+2=3
640 nm	25+27 (Ch.1+Ch.3)	1+4=5
510 nm + 640 nm	25+26+27 (Ch.1+Ch.2+Ch.3)	1+2+4=7

License

This open-source project is released under the CERN Open Hardware License. Our aims are to promote open scientific hardware development and to share our engineering solutions.