Confocal Microscopy

This system mates an upright microscope with four lasers: a UV Diode (405nm), an Argon laser (458nm, 476nm, 488nm, 514nm), a red Diode laser (543nm), and a HeNe laser (647nm). The system has a spectrophotometer scan head that allows the user to tune the four PMT detectors to any emission wavelength, and it allows simultaneous imaging with an additional transmitted light detector. It can also perform spectral scanning of fluorescence for generation of dye emission spectra and for separation of dyes with overlapping emission spectra. It also features a motorized stage that can mark and find multiple points and automate montage acquisition and stitching to generate high resolution images of large areas.

At left the Leica SP5 DM microscope. At right, image of Drosophila heart by Jianbo Na

Technical Specifications

Laser Lines

Detectors

Emission Filters

Dichroics

Scan Modes

Bit Depth

Resolution

405 Diode

4 PMTs

Tunable

RT
30/70

Line

8, 12, 16

max:

8192 x 8192

Argon (458, 476, 488, 514)

1 Trans PMT

 

DD 488/543

Frame

 

min: 16 x 16

HeNe 543

   

RSP 500

ROI

   

HeNe 633

   

Substrate

Spectral decon-volution

   
   

 

TD 488/543/633

Time lapse

   
   

 

DD 458/514

Mon-tage

   
       

FRAP

   
       

FRET – AB, SE

   

Objectives

Magnification

Immersion

NA

10x

Air

0.4

20x

Air

0.7

40x

Oil

1.25

63x

Oil

1.4

10x (dipping)

Water

0.3

20x (dipping)

Water

0.5

40x (dipping)

Water

0.8

User Protocol

This system has an inverted microscope enclosed within a CO2, humidity, and temperature-controlled chamber for live cell confocal imaging. It has four lasers: a UV Diode (405nm), an Argon laser (458nm, 476nm, 488nm, 514nm), a DPSS laser (561nm), and a HeNe laser (647nm). Its spectrophotometer scan head uses up to five PMT detectors to detect five different tunable emission wavelengths, allows simultaneous imaging with an additional transmitted light detector, and can perform multiple types of FRET imaging experiments. Like the DM upright confocal, it can also perform spectral scanning of fluorescence for generation of dye emission spectra and for separation of dyes with overlapping emission spectra. But to further distinguish it from the DM, it also has a “resonance scan” mode that enables up to 25 512x512 frames per second imaging for time lapse experiments. The motorized stage contains adapters for different dishes and chamber slides to accommodate a wide range of experimental setups. It also can mark and find multiple points and automate montage acquisition and stitching to generate high resolution images of large areas.

The Leica SP5 DMI microscope with environmental control

Technical Specifications

Laser Lines

Detectors

Emission Filters

Dichroics

Scan Mode

Bit Depth

Resolution

405 Diode

4 PMTs

Tunable

RT 30/70

Line

8, 12, 16

max: 8192 x 8192

Argon (458, 476, 488, 514)

1 Trans PMT

 

DD 488/561

Frame

 

min: 16 x 16

DPSS 561

1 HyD

 

RSP 500

ROI

   

HeNe 633

   

Substrate

Reson-ance

   
     

TD 488/561/633

Spectral decon-volution

   
     

DD 458/514

Time lapse

   
       

Montage

   
       

FRAP

   
       

FRET – AB, SE

   
       

Live Data

   

Objectives

Magnification

Immersion

NA

10x

Air

0.4

20x

Air

0.7

40x

Oil

1.25

63x

Oil

1.4

63x

Glycerin

1.3

100x

Oil

1.4

20x*

Multi

0.7

* available on request

User Protocol
Tiling Profocol

 

 

This state of the art laser scanning confocal microscope is equipped with Airyscan. Airyscanning dramatically increases signal by using an array detector to capture light that is otherwise rejected by the confocal pinhole. The extra light can be used to increase signal to noise (capturing at the same speed) or resolution (Capturing at a reduced speed). Airyscan is a super-resolution strategy: 1.7x the resolution of a standard confocal microscope in the xy plane (~140nm) and about 30% improvement in the z-axis: ~400nm. Airyscan can be used with all of the fluorophores that can be imaged with this confocal.

Zeiss LSM880 with Airyscan and environmental control

The LSM880 system sits on an inverted AxioObserver microscope stand and is enclosed by a C02, 02, humidity and temperature-controlled chamber for imaging living samples. It has seven laser lines (see below) and is equipped with two fluorescence PMTs, one PMT for transmitted light, and a highly sensitive GaAsP detector that can capture 32 channels simultaneously. Up to 13 images can be captured per second at 512 X 512 pixels.  Linear scanning makes this microscope suitable for a variety of quantitative image measurements such as fluorescence correlative spectroscopy. The stage is motorized permitting multidimensional time lapse imaging at several sites and tile scanning to generate high resolution images of large areas. For live imaging, stage inserts can accommodate 35mm, 60mm and Labtek/IBIDI chamber slides or multiwell glass bottom plates. Hardware is controlled by Zeiss Zen Black software, which also incorporates some image processing options.

Technical Specifications

Laser Lines (nm)

Detectors

Scan Mode

Bit Depth

Diode Laser 405 nm

32 channel GaAsP

line

8

Argon Laser 458/488/514nm

T-PMT (transmitted light)

frame

12

DPSS laser 561nm

2-PMT

 

16

HeNe laser 594/633 nm

Airyscan detector

 

 

Acousto-optical tunable filter

 

 

 

Objectives

Magnification

Immersion

N.A.

Working dist (mm)

10X

air

0.45

2.0

20X

air

0.8

0.55

40X

oil

1.4

0.2

40X*

water

1.2

0.28

63X

oil

1.4

0.17

100x

oil

1.46

0.1

* available upon request

User Protocol
Airyscan Protocol

The LSM780 system sits on an upright Axio Examiner microscope stand with a fixed stage. It has seven laser lines (see below) and is equipped with two fluorescence PMTs, one PMT for transmitted light, and a highly sensitive GaAsP detector that can capture 32 channels simultaneously. Up to 13 images can be captured per second at 512 X 512 pixels.  Linear scanning makes this microscope suitable for a variety of quantitative image measurements such as fluorescence correlative spectroscopy. Hardware is controlled by Zeiss Zen Black software, which also incorporates some image processing options. Microscope is enclosed by a C02, 02, humidity and temperature-controlled chamber for imaging living samples.

Zeiss LSM780 with a darkened environmental control box

Technical Specifications

Laser Lines (nm)

Detectors

Scan Mode

Bit Depth

Diode Laser 405 nm

32 channel GaAsP

line

8

Argon Laser 458/488/514nm

T-PMT (transmitted light)

frame

12

DPSS laser 561nm

2-PMT

 

16

HeNe laser 594/633 nm

Airyscan detector

 

 

Acousto-optical tunable filter

 

 

 

Objectives

Magnification

Immersion

N.A.

Working dist (mm)

10X

air

0.45

2.0

20X

air

0.8

0.55

40X

oil

1.4

0.2

40X

water

1.2

0.28

63X*

oil

1.4

0.17

100x*

oil

1.46

0.1

* available on request

User Protocol

 

This is a custom system comprised of a Zeiss Axio Observer Z1 inverted microscope, Yokogawa CSU-X1 spinning disk, and controlled by MetaMorph image acquisition software.  Its Prairie Technologies Aurora solid state diode laser launch has five laser lines: 405nm, 488nm, 515nm, 561nm, 640nm.  This microscope is ideal for long term, high speed, time lapse imaging.  It has multiple features to enable a wide range of live confocal imaging experiments, including:  a Tokai Hit enclosed stagetop incubator system with controlled temperature, CO2, and humidity, as well as lens heater; adapters for cell chambers, dishes, and slides; an encoded motorized stage that can mark multiple points within a sample or between different wells; a Piezo focus device for nanopositioning during high speed, high resolution z-stack imaging; and an on-site tissue culture facility to maintain cells prior to imaging.  For additional speed, the dual Hamamatsu EM CCD digital cameras enable simultaneous imaging of two fluorescent channels at a time.

Yokogawa spinning disk confocal microscope

Technical Specifications

Laser Lines (nm)

Cameras

Emission Filters

Dichroics

Scan Mode

Pixel Clock Rates

Resolution

405

Camera 1:

LP561

T405

Frame

Normal CCD:

max: 512 x 512

488

Hamamatsu

BP 452/45

488

z-stack

0.69 MHz

min: 128 x 128

515

EM-CCD

BP 542/45

568

Time-lapse

 

12 bits

561

C9100

BP 676/29

647

Multiple positions

EM-CCD:

 

640

 

BP 525/45

   

0.69 MHz

 
   

BP 605/15

   

2.75 MHz

 
 

 

     

11 MHz

 
 

Camera 2:

LP 405

T442

     
 

Hamamatsu

BP 452/45

514

     
 

EM-CCD

BP 525/30

647

     
 

C9100

BP 676/29

       
   

LP 561

       
   

BP 542/27

       

Objectives

Magnification

Immersion

NA

20x

Air

0.4

40x

Oil

0.6

40x

Oil

1.3

63x

Oil

1.4

100x

Oil

1.4

User Protocol
Dual Camera Protocol