2P calibration method from June 29, 2011

Loaded Alexa-568 solution (1uL of 5mM stock in 100uL of water) onto a haemocytometer and imaged using 810nm IR. 

Images were taken at zoom levels 1X, 2X, 3X, 5X, and 10X, each at 1024X1024, 512X512, and 256X256 resolutions.



Results:
at 1X zoom, 200umX200um = 590X600pixels, 295X300pixels, 147X150pixels at 1024, 512, 256 resolution respectively

at 2X zoom, 100umX100um = 591X601pixels, 295X300pixels, 148X150pixels at 1024, 512, 256 resolution respectively

at 3X zoom, 100umX100um = 890X901pixels, 445X451pixels, 222X224pixels at 1024, 512, 256 resolution respectively

at 5X zoom, 50umX50um = 749X749 pixels, 374X375pixels, 187X187pixels at 1024, 512, 256 resolution respectively

at 10X zoom no complete squares are visible

FOV:
1X: 
1024*(200/590)= 347.1um wide by 1024*(200/600)= 341.2um high
512*(200/295)= 347.1um wide by 512*(200/300)= 341.3um high
256*(200/147)= 348.3um wide by 256*(200/150)= 341.3um high
ratio: 1024*(200/590)/(1024*(200/600)) = 1.017


2X: 
1024*(100/591)= 173.3um wide by 1024*(100/601)= 170.4um high
512*(100/295)= 173.6um wide by 512*(100/300)= 170.7um high
256*(100/148)= 173.0um wide by 256*(100/150)= 170.7um high
ratio: 1024*(100/591)/(1024*(100/601)) = 1.017

3X: 
1024*(100/890)=115.1um wide by 1024*(100/901)= 113.7um high
512*(100/445)=115.1um wide by 512*(100/451)= 113.5um high
256*(100/222)=115.3um wide by 256*(100/224)= 114.3um high
ratio:1024*(100/890) /(1024*(100/901)) = 1.012

5X: 
1024*(50/749)=68.4um wide by 1024*(50/749)= 68.4um high
512*(50/374)=68.4um wide by 512*(50/375)= 63.3um high
256*(50/187)=63.4um wide by 256*(50/187)= 63.4um high
ratio: 1024*(50/749)/(1024*(50/749)) = 1.000