Drag and drop the sample H&E Kidney sample onto the microscope stage.

Light and Fluorescence microscopy includes a complex series of operational modes that you can select from the panel at the lower left. We suggest that you start with Set-up and Brightfield, but you are able to choose whichever mode you wish to explore. Samples and microscope structure will change to reflect the different applications of the various modes.

MICROSCOPE MODE

SETUP & BRIGHTFIELD

FLUORESCENCE

PHASE CONTRAST

DIC

POLARISED LIGHT

DARKFIELD

CONFOCAL

SUPER-RES – STED

SUPER-RES – SMLM

INSERT

PHASE

DIC FILTER

POLARISER/ANALYSER

DARKFIELD RING

HALOGEN LAMP

MERCURY LAMP

FOCUS

FIELD DIAPHRAGM

RIGHT EYEPIECE

OBJECTIVES

MOVE SLIDE

–

0

+

LIGHT INTENSITY

CONDENSER POSITIONING

CONDENSER FOCUS

CONDENSER APERTURE

LIGHT INTENSITY

CHANNEL 3
CHANNEL 4

MICROSCOPE MODE

SETUP & BRIGHTFIELD

FLUORESCENCE

PHASE CONTRAST

DIC

POLARISED LIGHT

DARK FIELD

CONFOCAL

SUPER-RES – STED

SUPER-RES – SMLM

CHANNEL 2

Ex 488 Em 500-550

100%

80V

60

0%

20V

-60

LASER

GAIN (HV)

OFFSET

CHANNEL 3

Ex 561 Em 570-600

100%

80V

60

0%

20V

-60

LASER

GAIN (HV)

OFFSET

CHANNEL 4

Ex 640 Em 650-700

100%

80V

60

0%

20V

-60

LASER

GAIN (HV)

OFFSET

Z-STACK & PINHOLE

TOP Z-POS

BOTTOM
Z-POS

no set

no set

OPTIMISED

RANGE

OPTICAL SECTION THICKNESS

LIVE VIEW

CAPTURE IMAGE

CAPTURE Z-STACK

SCAN RATE (FRAMES/SEC)

0.5

ZOOM

CHANNEL 1

MICROSCOPE MODE

SETUP & BRIGHTFIELD

FLUORESCENCE

PHASE CONTRAST

DIC

POLARISED LIGHT

DARK FIELD

CONFOCAL

SUPER-RES – STED

SUPER-RES – SMLM

CHANNEL 1

Ex 633 Em 690-720

LASER

STED LASER

775 nm

LASER

AUTOSCALE

DECONVOLUTION

LIVE VIEW

CAPTURE IMAGE

PIXEL SIZE

120 nm

ZOOM

1x

6x

CHANNEL 1

MICROSCOPE MODE

SETUP & BRIGHTFIELD

FLUORESCENCE

PHASE CONTRAST

DIC

POLARISED LIGHT

DARK FIELD

CONFOCAL

SUPER-RES – STED

SUPER-RES – SMLM

CHANNEL 1

Ex 647 Em 665-715

LASER

1000 µm

FOCUS

CAMERA SETTINGS

DATA ACQUISITION

ACTIVATION LASER

POWER

2%

DURATION

100

MS

EVERY

500

FRAMES

LIVE VIEW

CAPTURE IMAGE

CAPTURE SMLM IMAGE

DATA PROCESSING

INTENSITY THRESHOLD

LOW

HIGH

PROCESS SMLM IMAGE

DRIFT CORRECTION

IMAGING SOFTWARE

×

EXPOSURE TIME

0ms

8ms

HISTOGRAM

×

HISTOGRAM

×

×

ACTIVITIES MENU

VISUALISING YOUR SAMPLE

OBJECTIVE LENS

PINHOLE

PIXEL DWELL

PIXEL ARRAYS

MULTICHANNEL IMAGES

3D & Z-STACKS

Excitation–emission: example for viewing GFP

Click to see each channel and then combine the images into the overlay.

×

Click to see each channel and then combine the images into the overlay.

×

Note how much brighter and blurrier the open pinhole image is and how much extra light comes from out of focus planes when the pinhole isn’t blocking it. This is obvious based on number of oversaturated red pixels.

×
PINHOLE = 1.0 AU
PINHOLE = OPEN

Compare it with the images that you have just collected. If you close the pinhole too much, light can’t reach the detector.

×
PINHOLE = 0.4 AU (closed)
PINHOLE = OPEN
PINHOLE = 1.0 AU

At a Pixel Dwell Time of 12 µseconds (compared to a dwell time of 2.0 µseconds), the time to collect photons is increased by a factor of 6x. This means the image will be much BRIGHTER and the image scan rate will be much SLOWER. Most common dwell times are around the 2–5 µsecond range.

×
12 µsec
2 µsec
4 µsec

The resulting image is a merge of both AlexaFluor568 and AlexaFluor647.
Save this image to compare it to a sequentially acquired image.

×

The resulting image is a merge of both AlexaFluor568 and AlexaFluor647 but scanned sequentially.

×

Simultaneous

Sequential

×

Note that each image is acquired frame by frame to ensure the absence of the spectral bleedthrough of the 568 into Channel 4 that was present in the simultaneous image.

×

CONFOCAL

STED

×

Use the zoom buttons to have a closer look into a specific region and drag over the image to pan.
When ready close this window to continue.

Lower (6%) STED laser intensity

Higher (35%) STED laser intensity

Higher (35%) STED laser intensity
(with autoscale)

×

Use the zoom buttons to have a closer look into a specific region and drag over the image to pan.
When ready close this window to continue.

CHANNEL 4

Channel 3 3D image projection and rotation.

×

×

×

×

×

Please wait while the image scans

×

Diagram

×

Important Points:

×
  1. The plane of the optical section is the x,y plane.
  2. Moving the focus moves the sample stage in the z direction.
  3. The purpose of the confocal pinhole is to block out-of-focus light from reaching the detector.

×

Geological polarised microscopes have more control features to collect images like these.
In cross-polarised light, the Birefringence colour or shade changes as the mineral is rotated because the proportion of polarised light being conveyed along the mineral optic axes changes with reorientation of the mineral. Plagioclase or pyroxene appear black in cross-polarised light when one of their optic axes is aligned with polarised lighting. The striping in both pyroxene and plagioclase is due to twinning. Lamellar twinning is obvious in plagioclase, with repeated thinly spaced changes in birefringence due to stepped changes in crystallographic orientation. Simple twinning can also be seen in some plagioclase grains, where half of a mineral grain has one crystallographic orientation and the other half has another orientation.

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