The applications of fluorescence microscopy span medical diagnostics, bioengineering and biomaterial analytics. Full exploitation of fluorescent microscopy is hampered by imperfections in illumination, detection and filtering. Mainly, errors stem from deviations induced by real-world components inducing spatial or angular variations of propagation properties along the optical path, and they can be addressed through consistent and accurate calibration. For many applications, uniform signal to noise ratio (SNR) over the imaging area is required. Homogeneous SNR can be achieved by quantifying and compensating for the signal bias. We present a method to quantitatively characterize novel reference materials as a calibration reference for biomaterials analytics. The reference materials under investigation comprise thin layers of fluorophores embedded in polymer matrices. These layers are highly homogeneous in their fluorescence response, where cumulative variations do not exceed 1% over the field of view (1.5 x 1.1 mm). An automated and reproducible measurement methodology, enabling sufficient correction for measurement artefacts, is reported. The measurement setup is equipped with an autofocus system, ensuring that the measured film quality is not artificially increased by out-of-focus reduction of the system modulation transfer function. The quantitative characterization method is suitable for analysis of modified bio-materials, especially through patterned protein decoration. The imaging method presented here can be used to statistically analyze protein patterns, thereby increasing both precision and throughput. Further, the method can be developed to include a reference emitter and detector pair on the image surface of the reference object, in order to provide traceable measurements.
Calibration and validation of fluorescence microscopy devices and components require a high level of stability and repeatability in their fluorescent properties, both spatially and temporally. In order to establish a dependable reference point, from which all variations within the microscope and peripheral devices can be tested, an exceedingly homogeneous fluorescence response must be provided through a calibration tool. We present material system optimization and microfabrication process development, as well as long-term stability considerations for such a calibration tool. Stringent specifications for film thickness (< 1μm ± 0.1% over 1.5x1.5 mm) and for fluorescence response distribution (within 1%) apply, and should hold for up to 100 hours under continuous white irradiation. Low conversion efficiency demands high pick up efficiency and therefor reduces focal depth by high NA of applied fluorescence microscope lens. High spatial resolutions demands use of high quality lenses that typically show low field curvatures and good chromatic corrections. Therefore, the focal plane is flat and well defined in the z-plane.
Fluorescent, ligand capped core-shell quantum dots (SMQDs) were embedded in diluted PMMA at low concentrations. The formulations were spin-coated on silicon and glass wafers to obtain films with thicknesses under 1 μm and low variations on a 100 mm wafer. Fluorescence properties of the SMQD were preserved in the matrix material, and agglomerations were not detectable in the fluorescence response nor in SEM images. Gradual degradation of the fluorescence response due to film aging was managed through robust packaging solutions.
In recent years, many compact spectrometers for purposes such as environmental monitoring and process quality control
in industrial production have been realized. However, most of them still employ spectrometer mounts with focal lengths
in the range of several cm. Therefore, their size is about that of a palm which is too large for OEM-use in handily sized
optical sensor equipment.
Accordingly, we have developed a thumb-sized, truly miniaturized spectrometer for the spectral range 340nm to 750nm,
which is particularly suited for use inside hand-held or portable color management sensor equipment. The spectrometer
is using a self-imaging, aberration-corrected concave grating with very short focal length and a blazed grating profile for
high diffraction efficiency. The grating is replicated onto the top of a convex glass lens using nano-imprint technology.
Opposite to the concave grating, a dedicated C-MOS image sensor with an in-built on-chip slit is placed. The slit with a
width of 75μm is formed into the silicon chip using MEMS technology. Due to this advanced technology, the distance
between the sensor area and the slit is as small as 1mm. Based on this high level of integration, the number of optical
components could be kept to a minimum and the distance between the concave grating and C-MOS image sensor is
about 8.5mm only.
In summary, we have realized a well-performing miniaturized spectrometer with an extremely small package size of
28mm - 17mm - 13mm and a weight of only about 9g, which is highly suited for integration into optical sensing