Imaging Technology

Our lab designs and builds all of its own imaging systems. This allows us to create highly customized configurations allowing us to explore in-vivo phenomena using light. This page provides details of our systems, including free software downloads.

The systems described below include:

1) In-vivo two-photon microscope / dual beam methods

2) Multispectral optical intrinsic signal imaging (MS-OISI)

3) Laminar Optical Tomography (LOT)

4) Swept Confocally-Aligned Planar Excitation (SCAPE) microscopy formerly known as Laser-Scanning Intersecting Plane Tomography (L-SIPT))

 

In-vivo two-photon microscope / dual beam methods

Our two-photon microscope design is optimized for in-vivo imaging. Its three emission channels and flexible fliter configuration allow imaging of almost any source of contrast including second harmonic generation, intrinsic fluorescence, fluorescent proteins, active dyes and conventional fluorescent stains. 3 or more sources of contrast can be distinguished and imaged in paralell by the system at up to 30 frames per second, to depths exceeding 500 microns in living tissues. The system is fully automated with all stages and instrument settings controllable via a Matlab graphical user interface (GUI). This software also controls the system's two Spectra Physics MaiTai Ti:Sapphire lasers, allowing synchronous wavelength scanning and imaging allowing hyperspectral microscopy (for more details see here).

The recent addition of a second (MaiTai DeepSee) laser to this system has turned it into a powerful dual-beam platform allowing a host of new techniques to be incorporated. Beams can be re-routed to a high-speed resonant scanner for faster acquisition, or to our spatial light modulator for 3D beam patterning or adaptive optics correction. We are also exploring techniques that illuminate the tissue with both beams simultaneously for parallel imaging of two locations, or for photoactivation for so-called 'Interventional Microscopy'.

in-vivo tow-photon microscope design

Figure 1. Our in-vivo two-photon microscopy system was designed to allow high-speed imaging of 3 or more sources of contrast in living tissue. The system can also perform hyperspectral microscopy. For more information see Radosevich et al 2008, Hillman 2007 and Bouchard et al 2006.

in-vivo imaginge of vasculature and astrocytes

Figure 2. 3D images of vasculature (dextran conjugated fluorescein) and astrocytes (SR101-stained) in-vivo. Each microscope channel can be anaylsed independently, or together. See here for more information on our brain imaging projects.

 

 

Multispectral Optical Intrinsic Signal Imaging (MS-OISI)

Camera-based spectroscopic optical imaging systems can map functional parameters of living systems by monitoring changes in absorption and/or fluorescence contrast. These systems have been used to study biological questions ranging from cortical hemodynamics to more recent applications in dynamic molecular imaging of pharmacokinetics. We are have developed several high-speed CCD-camera based spectroscopic optical imaging systems that are not hindered by the image acquisition rate limits which have typically constrained spectroscopic imaging systems. This is acheived by free-running the system's camera at its maximum rate, and using its electronic exposure signal to generate a drive waveform for a sequence of different high power, appropriately filtered light emitting diodes (LEDs). Our new high-speed systems are capable of imaging both absorption and fluorescence contrast in parallel at rates exceeding 100 frames per second and greater than 1 megapixel spatial resolution. With appropriate optics, our systems can image living systems ranging in size from small animals to single cells.

Our first system, based on the Dalsa 1M60 camera has provided multi-spectral optical intrinsic signal imaging (MS-OISI) data on exposed rodent cortex in a number of studies, as well as dynamic contrast enhanced small animal molecular imaging data (DyCE). We have since developed a low cost, portable version of our system which can be readily assembled from an inexpensive firewire camera, laptop, LEDs and an Arduino microcontroller for a total cost of less than $5,000. We are happy to provide instructions to assemble this system in addition to providing free download of our control software SPLASSH (SPectral Light Acquisition Software for Signaling and Hemodynamic imaging). For more details on this system, see Sun et al. For software download and instructions, please click here.

SPLASSH imaging system diagram

Figure 3. Schematic of our fast camera system showing main components and signal flow. See Sun et al for more dtails.


So-called 'Optical Intrinsic signal imaging' (OISI) has been used for over 20 years to examine cortical responses to stimulus. In its simplest form, such imaging requires only a light source at a specific wavelength, and a camera to detect changes in the amount of light remitted from the brain's surface. However, it is now widely understood that changes in reflectance of the brain are due to changes in the local concentration of absorbing oxy- and deoxy-hemoglobin. It is therefore possible to make OISI more quantitative if multiple wavelengths of illumination are used, and modeling of light propagation is employed to convert measurements of light intensity into changes in oxy- and deoxy-hemoglobin. A further extension of this kind of imaging is to add fluorescent voltage or calcium sensitive dyes to the brain to allow equivalent imaging of neuronal activity. See Hillman, 2007 and Bouchard et al, 2009 for more details.

MS-OISI example

Figure 4. Multispectral optical intrinsic signal imaging (MS-OISI) data acquired on exposed rat somatosensory cortex during 4 second electrical hind-paw stimulation. Raw images acquired at interleaved blue (460 nm) and green (530 nm) illuminations were used to convert to maps of changing oxy-, deoxy- and total hemoglobin concentrations (HbO2, HbR and HbT). See Bouchard et al for more details.

 

 

Laminar Optical Tomography (LOT)

LOT uses laser-scanning instrumentation similar to a confocal microscope. Light is serially injected into the surface of a tissue. Once in tissue, light will undergo scattering events, encountering absorbers and fluorophores along its path. Some of this backscattered light will be remitted from the tissue surface at different distances from the source position. Light emitting further from the incident source has, on average, travelled more deeply into the tissue. LOT therefore acquires depth sensitive information by measuring off axis backscattered light. Whereas a confocal microscope rejects off axis backscattered light with a pinhole, LOT replaces the pinhole with a slit and uses an array of optical detectors to acquire depth sensitive tomographic measurements. The three-dimensional distribution of optical contrast represented by these measurements can be deduced using models of light propagation, and a tomographic style image reconstruction.

 

Multispectral LOT

Figure 1. Laminar Optical Tomography is capable of 3D imaging of absorption at three wavelengths and fluorescence simultaneously at frame rates of over 100Hz. For more information see Burgess et al 2008 and Yuan et al 2009.

 

We have applied LOT to a range of living tissues including the brain, heart and skin. For more information on applications of LOT see Hillman et al 2007, Hillman et al 2007b, Yuan et al, 2009, Hillman and Burgess 2009, Burgess and Hillman 2010 and Burgess et al 2010.

 

LOT of Brain

Figure 2. Photograph and technical layout of our current laminar optical tomography system from Yuan et al 2009.

 

Laminar Optical Tomography can add a depth-dimension to conventional multi-spectral optical brain imaging. LOT can currently image hemodynamic activity to > 2mm, with 100-200 micron resolution at over 80 frames per second.

To date, we have demonstrated that LOT can resolve the oxy– and deoxy-hemoglobin responses in individual vascular compartments in the somatosensory cortex during forepaw stimulation. This was achieved using a unique spatiotemporal linear fitting procedure based on the characteristic temporal behavior of the arteriolar, capillary and venous responses. We have now added fluorescence imaging capabilities to LOT to allow simultaneous depth-resolved imaging of voltage or calcium sensitive fluorescent dyes to enable 3D studies of neurovascular coupling in the living brain through the full thickness of the cortex. See Hillman et al, 2007 for more details.

LOT of Brain

Figure 4. Laminar Optical Tomography of the hemodynamic response to forepaw stimulus in the rodent somatosensory cortex (thinned skull). For more details see Hillman et al 2007.

 

 


 

Funding
National Institute of Neurological Disorders and Stroke (R21 NS053684 PI: Hillman and R01 NS063226 PI: Hillman), NSF Career Award (0954796 PI: Hillman), NSF IDBR (1063315 PI: Shepard)

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