Confocal Microscopy

Equipment

Leica TCS SP8 with five lasers

The Leica TCS SP8 sits on an inverted Leica DMi8 microscope stand and has 5 lasers: a UV Diode (405nm), an Argon laser (458nm, 488nm, 514nm), a 561nm, a 594nm and a 633nm. Its spectrophotometer scan head is comprised of 1 PMT and 3 HyD detectors (more sensitive for weaker signals) to detect five different tunable emission wavelengths, as well as a transmitted light detector for the addition of DIC imaging. The motorized stage and “Navigator” software package allow the user to map large sample areas at high resolution in addition to imaging multiple, non-contiguous single positions. Full OkoLab incubation is available as well for live-cell experiments. Hardware is controlled by Leica LAS X software. Leica’s “Lightning” mode is also available, allowing for 40 frames-per-second multi-color imaging at 512x512 pixels, down to 120nm lateral resolution

Objectives

1) HC PlanApo 10x/0.4 "A"

Requires 0.17mm coverglass

air/dry

 

2) HC PlanApo 20x/0.75 "C"

Requires 0.17mm coverglass

air/dry

 

3) HC PlanApo 40x/1.3 "D"

Requires 0.17mm coverglass

oil immersion

 

4) HC PlanApo 63x/1.4 "E"

Requires 0.17mm coverglass

oil immersion

 

5) HC PlanApo 100x/1.4 "D"

Requires 0.17mm coverglass

oil immersion

 

6) (none)

 

 

 

 

 

 

Excitation

405nm

 

 

 

458nm

 

 

 

488nm

 

 

 

514nm

 

 

 

561nm

 

 

 

594nm

 

 

 

633nm

 

 

 

 

 

 

Detectors

3x HyD

Frequency Range: 10-1800Hz

 

 

1x PMT

Detection Range: 400-800nm

 

 

1x T-PMT (for transmitted light)

 

 

 

 

 

 

Dichroics

458/514/561

 

 

 

488/561/633

 

 

 

458/514

 

 

 

488/594

 

 

 

RT 15/85

 

 

 

 

 

 

Compatible with:

standard slides (76x26mm)

 

 

 

standard well plates

 

 

 

 

 

 

Accessories

Partial environmental controls

 

 

 

Lightning mode - 40FPS @ 512x512; 120nm XY resolution

 

 

Long a workhorse of the Microscopy CoRE, the Leica SP5 is a seven-laser line inverted imaging system with many capabilities. The Leica SP5 features six detectors for simultaneous imaging (4 PMT, 1 HyD, 1 T-PMT); a high-speed resonance scanner for up to 25 frames-per-second at 512x512 resolution; a full temperature- and CO2-controlled incubation chamber; the ability to perform multiple types of FRET imaging; and the ability to perform spectral scanning in order to accurately separate overlapping emission spectra. A motorized XY stage easily adapts to different sizes of dishes, chamber slides and standard glass slides. The LAS AF software suite makes setup and image acquisition easy while giving users the opportunity to expand on their imaging capabilities as their needs evolve.

Computer room

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

 

 

Building on the powerful Leica SP8 confocal scanhead, this super-resolution system features three separate additional lasers for Stimulated Emission Depletion (STED) across a range of florophores. The STED 3x setup provides XY resolution down to 50 nanometers and improved Point Spread Functions for optimal resolution with any sample.

In addition to five standard laser lines and five detectors (2 PMT, 3 HyD), the STED system has a pulsed tunable White Light Laser that will excite fluorophores from 470-670nm. The WLL gives users the ability to precisely maximize excitation efficiency of one fluorophore at a time in multiplexed samples while also enabling the use of advanced imaging techniques such as FLIM, which requires a pulsed laser. The pulsed WLL coupled with the pulsed STED 775nm laser line even allows for FLIM STED, a super-resolution technique that permits separation of emission signals based on temporal data. "Tau STED,” the latest addition to this system, aids researchers with time-correlated photon counting, giving users the unique ability to separate fluorescence signals based on time-of-detection and the actual number of photons collected. Availability of Phasor Plot analysis tool with the module enables effective FLIM and FRET-FLIM studies.

The LAS X software suite from Leica simplifies setup for even the most complex setups.

Inverted microscope system

Technical Specifications

Laser Lines (nm)

Detectors

Scan Mode

Bit Depth

Resolution

Diode Laser 405 nm

2 PMT

Line

8

max: 8192 x 8192

Argon Laser (458, 476, 488, 514)

3 HyD

Frame

12

min: 16 x 16

White Light Laser (WLL) 470-670nm

T-PMT (transmitted light)

STED

 

 

 

 

ROI

 

 

 

 

Spectral decon-volution

 

 

 

 

Time lapse

 

 

 

 

Montage

 

 


Objectives

Magnification

Immersion

NA

10x

Air

0.4

20x

Air

0.75

40x*

Oil

1.3

63x

Oil

1.4

93x **

Glycerin

1.3

100x **

Oil

1.4

* available on request

** suitable for STED imaging

 

Harnessing the power of a 32-channel, honeycomb-shaped array detector, the LSM880 with Airyscan offers super-resolution microscopy without the complications of restricted sample preparation or fluorophore choice - if your sample is compatible with a standard confocal microscope, it is compatible with the super-resolution Airyscan detector as well. Resolution improvements up to 140nm in XY and 400nm in Z are possible in combination with imaging speeds of up to 13 frames per second at 512x512 resolution.

The LSM 880 with Airyscan is built on the inverted Axio Observer.Z1 microscope and features a full incubation suite for temperature and CO2 control, making it ideal for live-cell studies. Multispectral imaging is also possible with the LSM880's native 32-channel Quasar detector and two additional PMT detectors: the entire spectrum is scanned at once to quickly build a database of relative fluorophore signal contribution from control samples, and the system can then easily separate emission signals that exhibit high spectral overlap during live imaging of multiplexed samples. DIC imaging is also possible through the use of a Transmitted PMT detector.

Zeiss LSM880 with Airyscan and environmental control

Zeiss LSM880 with Airyscan and environmental control

The LSM880 with Airyscan runs on ZEISS' ZEN black software platform. The software incorporates Tile Scan, Z-stack, Time Lapse and Bleaching functions for multidimensional experiments.

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

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

 

The CoRE's solution when optical sectioning and high-speed imaging are jointly required is the Yokogawa Spinning Disk Confocal. Built on the inverted ZEISS Axio Observer.Z1 frame and controlled by MetaMorph, the "SD" system is a powerful tool for observing fast live-cell phenomena where cells may not grow in a monolayer but where individual cell responses are needed, such as during drug treatments involving real-time injections. Illumination is provided by the Vortran VersaLase laser launch, with six excitation lasers (405/445/488/514/561/642nm) for multi-color imaging. A motorized stage provides opportunities to image multiple areas of a sample automatically over time. Stage-top incubation for short-term maintenance of cell viability is also available; features include a heated stage insert with cover, humidity and CO2 control, and an objective heater ring. Imaging is provided by the Yokogawa CSU-X1 spinning disk module and two Hamamatsu ImagEM EMCCD cameras for better low-light, rapid imaging; up to two fluorescent channels can be imaged simultaneously. On-site tissue culture equipment is also available, should researchers require it. This system also doubles as a conventional widefield microscope when coupled with the PCO.edge on the left camera port

Yokogawa spinning disk confocal microscope

Yokogawa spinning disk confocal microscope

Objectives

Magnification

Immersion

NA

20x

Air

0.4

40x

Air

0.6

40x

Oil

1.3

63x

Oil

1.4

100x

Oil

1.4