Microscopy - L4

usage of the emission pinhole

open/closing adds an extra axial layer z

in confocal microscopy single point at a time

2D-movement of whole specimen too slow and vibration-sensitive

flying spot by rotating mirror -> later pair of oscillating mirrors

important parts: laser, X- and Y-mirrors, specimen, light, both chromatic beam splitters, both pinholes, both detectors (PMTs)

filter cube

• wavelength of dichroic mirrors in excitation/emission light filters

• 300-900 nm

dwell time

• time the laser spot remains in one location

• increase -> improve signal to noise ration, but bleaching

pinhole

• key determinante

• restricts light getting to sensor (not specimen)

• size reduction -> better exclusion of out-of-focus light, but intensity reduction

imaging speed

• two oscillating mirrors moving at different rates

• X-axis: travels across image -> fast

• Y-axis: travels small distances -> slow

photomultiplier

• most common detection devise

• must be cooled: high temperature -> increased noise

• low gain -> not all fluorescence detected

• high gain -> output signal saturated

light sources

• lasers of 405-635 nm

• suited for fluorescnce imaging

optical sectioning

• parts above/below imaging point illuminated weakly -> z-stack

• assembled into 3D later

detector consists of 32 elements, structured in a hexagonal pattern

each elements detects the same point in a different manner

single images are combined later

higher light yield, better resolution, less light intensity necessary

rotating disk of 1000s of pinholes

usage of many pinholes at the same time -> not one per image

detector consists of 32 elements in a hexagonal pattern

all elements detect the same point, but in different manner