SAIP - OPTICAL IMAGING

Optical imaging is a highly informative and flexible method of in vivo imaging.  It can be divided into two types:  Bioluminescence and Fluorescence Imaging.

Bioluminescence Imaging (BLI)
Bioluminescence or BLI is a powerful imaging technique that takes advantage of the insertion of a reporter gene (e.g. luciferase or LUC) into a viral vector (e.g. adenovirus, lentivirus).  Cells transfected with LUC express luciferase and can be grown and injected into animals.  When the substrate of luciferase, luciferin, is injected into the cell-bearing living animals, light will be produced by the cells in the presence of oxygen.  More sophisticated constructs can be made in which the BLI is conditional on specific substrates.  Whole mice BLI models have also been created. 

The BLI image is superimposed upon the white light image to create a typical BLI image (see below).  The device used to capture such images is called the Xenogen SPECTRUM (Caliper Life Sciences, Hopkinton, MA); scanner is pictured below.  The Xenogen SPECTRUM is straightforward to operate after brief training (provided by the SAIP staff) and should be used by the investigator with technical support provided by the SAIP-F staff.

The strengths BLI are high photon sensitivity and throughput.  Disadvantages of BLI are that it requires living intact animals (BLI cannot be used for post mortem imaging), the luciferin is relatively costly and time dependent and time must be taken to develop a stable transfected cell line.  For most optical imaging techniques a big disadvantage is that tissue absorption of light means that the only light that is seen comes preferentially from superficial structures.


Xenogen camera (IVIS SPECTRUM)

B16 melanoma cells expressing LUC were injected into the lower extremities of three mice scanned at different times after injection (1 day, 4 days and 12 days).  Note that inguinal adenopathy appears in last animal indicating metastases have occurred.

Fluorescence Imaging
Fluorescence imaging (FI) differs from BLI in that an excitation light source is required in order to detect the emission of light. Typically, a filtered excitation light source is used to excite a fluorophore which emits light at a higher wavelength.  There are two types of fluorohores: endogenous (e.g. GFP, RFP) and exogenous (eg. FITC, Rhodamine, Cy5.5).  Like BLI, endogenous imaging requires a transfected cell line.  Endogenous imaging requires the transfection of a cell line with a gene that produces a fluorescent protein like GFP.  A significant technical challenge is presented by autofluorescence present in normal tissues.  Many key molecules in the body such as NADH, collagen among others are fluorescent and will interfere with the detection of the desired signal.  This results in lower contrast and hence, sensitivity, in FI compared to BLI. .  A method of improving the constrast and sensitivity of FI is offered by a spectral camera known as the MAESTRO (CRi, Woburn, MA).  Using a tunable LCD filter, this camera obtains images at multiple wavelengths to obtain the emission spectra at each pixel. The resulting three-dimensional image cube is then analyzed with the help of the user to separate the different autoflurescence from the fluorophores through a process called “unmixing”. With this method, it is possible to unmix the multiple fluorophores that are present and at the same time.

Maestro CRi unit consists of a light box, filtered light source and a filtered camera.   The animal is placed on the lighted stage and spectral images are obtained.

Exogenous Fluorescence Imaging
It is also possible to inject exogenous Fluorophores which can be targeted to specific cell types.  This does not require the creation of a cell line; the native cell line can be used, however, it does require the chemical synthesis of the targeted ligand-fluorophore conjugate. FI may be particularly useful in studying real time changes in fluorescence and will have clinical importance in surgery and endoscopy.  Below is an example of an exogenous fluorophore used in a mouse model of ovarian cancer.

A comparison of three animal mesenteries containing ovarian cancer metastases (SHIN3 model) using 3 different conjugated fluorophores.  Galactosamin Rhodamine Green (GSA Rhod G) brightly enhances the peritoneal metastases in this mouse model of ovarian cancer, whereas avidin Rhodamine Green (Av RhodG) and Bovine serum albumin Rhodamine green (BSA-RhodG) are taken up by tumors much less strongly.