Integrated Raman and Angular-Scattering Microscopy

Raman spectroscopy answers the question: what types of chemicals are in my sample?

Angular scattering answers the question: what's the size distribution of objects within my sample?

What's Been Done:

We have constructed a microscope that allows us to perform both types of measurements on a single cell.

This publication in Applied Optics shows how the IRAM instrument can determine chemical and size information from (a) mixtures of polystyrene beads that are organelle-sized, and (b) single immune cells of two types (neutrophils and lymphocytes). In the case of cells, we see differences between the two cells both chemically (the lymphocytes have more nuclear material) and morphologically (the granule population in neutrophils have a larger mean diameter (near 700 nm) than the lysozomes in lymphocytes (near 400 nm)). These differences correspond to what would be expected based upon electron microscopy.

IRAM has also been applied to identify single immune cells that have responded to the presence on an antigen. Activated CD8+ T cells show both chemical and morphological differences from their unactivated counterparts. The details of this study can be found in this publication in the Journal of Biomedical Optics.

"Changing IRAM to iRAM" -- The original IRAM system had some limitations. Changing between Raman and angular scattering acquisition modes was very time-consuming (sometimes requiring nearly an hour of alignment). The stage was moved by hand and provided no way of returning to a previously measured cell.

Through various system upgrades, both of these problems have been solved. The acquisition mode can be switched from Raman to angular scattering and back again in just a few seconds. A new computer-controlled stage has the ability to remember cell locations and return to them for repeated measurements.

These changes enable iRAM to track the molecular content (Raman spectroscopy), organelle size (angular scattering), and appearance (traditional microscope illumination) of several cells over time. Each of these types of data can be measured at several time points as the cells progress through a stimulated process.

Problem: Speckle in Angular Scattering Measurements

Angular scattering is used to study the sub-cellular structure of many types of cells, but rarely one cell at a time. Measuring the angular scattering from a single cell brings a new set of challenges. The scattering signals from various organelles interfere with each other and cause speckle. When measuring the scattering from many cells or whole tissues, the relatively large area contributing to the scattering signal causes the speckle grain size to be very small (J. W. Goodman, Laser Speckle and Related Phenomena). When only a single cell contributes to the scattering signal, the area is smaller and the speckle grain size is larger. This speckle pattern causes IRAM's size extractions to be unstable.

Solution: Interferometric Measurements

To overcome the limitations of speckle, we have added a reference beam to the angular scattering instrument. This allows us to extract the full complex field of the scattered light. The data can be analyzed by one of three methods: 1) isolating the signal from individual scatterers, 2) incorporating speckle into the forward model based on extracted scatterer locations, or 3) reducing the speckle by synthetically enlarging the illuminated region. These methods are currently being refined to overcome the effects of speckle and increase the stability of size extractions by angular scattering measurements.

What's Next?

Once the angular scattering system have been improved, it will be combined with the Raman spectrometer to once again take full IRAM measurements. With a stable, automated system, IRAM will be able to repeatedly measure single cells as they change over time. Possible biological applications include photo-dynamically treated cancer cells, platelets during activation and/or degranulation, or immune cells after exposure to an antigen.