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BIOIMAGING FACILITY

Director: James Keen, PhD Tel: (215) 503-8982
Location: 914 BLSB 233 South 10th Street Philadelphia, PA 19107	Contact: Wojciech Jankowski Tel: (215) 503-4770 Bioimaging Reservation System

The Bioimaging Facility makes every effort to support the greatest possible array of user requirements. One of the most basic of those is the need to be flexible with fluorescent dyes users are capable of using. To this end the facility microscopes can provide a variety of excitation and emission filters to cover a broad spectrum of fluorophores.

The Live Cell System

The Live Cell System is a traditional widefield fluorescence microscope that relies on filters to allow or block wavelengths on both the emission and excitation ends. The Live Cell System is powered by a broad spectrum Xenon Lamp that provides an even spectrum of excitation from around 380nm to 650nm. On the microscope itself we have a series of band pass filters that select excitation/emission pairs best used for the most common fluorophores. In addition, we have a fast-wavelength changer that can be used in conjunction with some of our di-chroic filters to allow quick sequential imaging of multiple fluorophores.

Filters Available for the Live Cell System

Filter Cube	Excitation Paths	Detection Paths	Notes
FITC Single Cube	D480/30	D535/40	FITC Filter in the Zeiss Microscope
TRITC Single Cube	D540/25	D605/55	TRITC Filter in the Zeiss Microscope
Cy5 Single Cube	HQ620/60	HQ700/75	Cy5 Filter in the Zeiss Microscope
Triple Cube	S395/10x, S484/15x, S555/25x	D450/25, D525/15, D580LP	Triple Filter in Zeiss Scope, use with excitation filters in the fast wavelength changer, can view Blue (DAPI), Green (FITC), or Red (Rhodamine)
GFP Single Cube	D425/40	D505/40	GFP Filter for Zeiss Microscope, ask to have it inserted
GFP/Red Double Cube	S485/15x, S555/25x	D520/20, D620/30	Double Filter in Zeiss Scope, use with excitation filters in the fast wavelength changer, can view Green Fluorescent Protein and dsRed
CFP/YFP Double Cube	S395/10x, S484/15x	D455/15, D545/25	Double Filter in Zeiss Scope, use with excitation filters in the fast wavelength changer, can view Cyan Fluorescent Protein and Yellow Fluorescent Protein

Confocal System

Unlike the Live Cell System, the Confocal Microscope is uses a series of LASERs to excite samples at very specific wavelengths, and has a wide array of emission filters that allow you personalize collection wavelengths to a greater degree, so much so that listing them all here is not reasonable. It is sufficient to say that with the Zeiss LSM 510 META Confocal, that as long as we have a LASER line that excites the fluorophore(s), there is a a filter combination or setting we can use to collect the light you want. Also, since the Confocal uses separate detectors to collect different channels, multiple color samples can be used simultaneously (the Live Cell system can not take images of multiple colors simultaneously because it uses a single black and white camera for detection), or in quick succession.

Below is a table of the Laser Lines the Confocal uses.

LSM 510 META Excitation Lines

Laser Name	Laser Excitation	Common Use
Diode Laser	405nm	DAPI
Argon Laser	458nm	Acridine Orange + RNA
	477nm	Fluo-3
	488nm	FITC, GFP
	514nm	YFP, EtBr + DNA
He/Ne Laser 1	543nm	TRITC, Rhodamine, Texas Red
He/Ne Laser 2	633nm	Cy5

In Vivo Capabilities

The LSM 510 Meta Confocal Microscope System is equipped with a temperature controlled sample chamber for live-cell imaging. The confocal system can perform Fluorescence Recovery After Photo-bleaching (FRAP) for live-cell protein dynamics experiments and Fluorescence Resonance Energy Transfer (FRET) for protein interaction studies. This system can image up to six fluorescence signals simultaneously, with fully programmable multi-location, time lapse, and z-stack scanning capability. The wide-field fluorescence microscope system is equipped with an incubated, CO2-controlled, and humidified chamber for overnight live cell imaging. It has a Lamda 64 fast-wavelength chamber to enable rapid sequential imaging of up to three fluorophores. Both systems are built on isolation tables to dampen vibration and are capable of auto-refocusing to counter drift. A programmable pump and perfusion chamber is available for use with either microscope system to allow media exchange and reagent addition during time-lapsed sample monitoring.