A Retrofitted Metallurgical Microscope Using Light Emitting Diodes for Multi-Spectral Imaging

Multi-spectral imaging (MSI) has made diagnosis of microscopic samples considerably easier and information abound. Most MSI systems use continuum light sources and filters for imaging purposes. However, these light sources and filters are relatively expensive, unstable due to extreme pressure and temperature and associated with prolong acquisition time. In this work, we present a metallurgical microscope retrofitted with light-emitting diodes (LEDs) as illumination sources for MSI microscopy. This multispectral LED imaging microscope (MSLEDIM) is relatively cheaper and capable of acquiring images in reflection, transmission and scattering modes at thirteen (13) different wavelengths ranging from ultraviolet to near infrared. The microscope has been demonstrated in biomedical and entomological research fields. The MSLEDIM can be used in various scientific research fields for imaging microscopic samples.

For years, most MSI systems use broad light sources and filters for microscopy.This is done by selectively transmitting and imaging light of different wavelengths from the broad light source of the microscope.During MSI application in microscopy, the selective transmission of light with different wavelengths by the filters depends on the stability of the spectrum of light source used and the time required during acquisition.However, the spectra produced by most light sources used in MSI microscopy are unstable due to extreme pressure and temperature.The filters used may also degrade with time.Besides, there is very little information about the chemical nature of the investigated samples when using the filters due to lack of spectral content in the images.More so, the light sources have limited number of spectral bands with relatively long acquisition time.This introduces uncertainties which lead to sample photo-bleaching and photo-kinetics, which may cause non-linear absorption and fluorescence (Brydegaard et al., 2011).
Light-emitting diodes (LEDs) as illumination light sources in MSI microscopy have made imaging easier and cost effective (Schubert, 2006;Brydegaard et al., 2011).Also, the stability of spectrum has been improved; modulation speed has increased to the sub-nanosecond scale and with a major reduction in losses during illumination (Herman et al., 2001;Cole & Turner, 2008;PicoQuant, 2011).LED Wavelengths currently available range from 240 nm to 7 μm (Roithner Laser Techniques, 2011).
In this work, we present a retrofitted metallurgical microscope which uses nine (9) LEDs emitting a total of 13 wavelengths ranging from 375 nm to 940 nm.These are used as illumination light sources for MSI in reflection, transmission and scattering modes.

Instrumentation
Components used for retrofitting the metallurgical microscope are classified into three main categories, namely: mechanical, optical and optoelectronic components.

Mechanical Components
The mechanical components of the MSLEDIM are the main metallic frame of the metallurgical microscope (SP-80, Brunel Microscopes Limited, 2009), three plastic holders for holding LED chips and a brass tube.The plastic holders have a heat sink, locking ring, and contact carrier assembly.The brass tube is made from brass chemically treated to remove its reflective surface.

Optical Components
A fibre ring (FB), consisting of a number of fibres bundled together and guided from a glass-polished end-face into anodized, aluminum housing, is used to diffuse the light in 360° around the central axis.An optical objective (Reflx™, Edmund Optics Inc.) based on reflective optics, with amagnification factor x15 and numerical aperture (NA) 0.28, is used for collecting and focusing of the light.This objective produces superior quality images over a broad spectral range.An optical diffuser (Edmund OpticsInc.) is used to achieve Lambertian distribution of light.Three (3) sets of 9 LEDs each were used as illumination light sources in reflection, transmission and scattering modes respectively.One of the LEDs emit 3 central wavelengths; 470 nm, 525 nm and 810 nm, while 2 others emit 2 central wavelengths each; 375 nm and 625 nm, and 400 nm and 850 nm respectively.Six LEDs emit single wavelengths at 435 nm, 590 nm, 660 nm, 700 nm, 750 nm and 940 nm.

Optoelectronic Components
A complementary metal-oxide-semiconductor (CMOS) imager (Guppy F-503B, NT63-371, Allied Vision Technologies © ), with a resolution of 2592x1944 pixels was used for image acquisition.The power of the CMOS imager is supplied via IEEE-1394 cable by a laptop computer (PC).National instrument data acquisition device (NI-DAQ), (model USB-6009, National Instruments © , 2009) was used to bridge the computer and switching of the LEDs.The DAQ has the ability to display both analogue and digital signals between 0-5 V in several channels and also the ability to retrieve an analogue signal.

Assembling
Figure 1 shows a schematic diagram of a retrofitted MSLEDIM.The LEDs were arranged and soldered unto 3 stripboards (LED mounts) to serve as the illumination light sources for reflection (M1), transmission (M2) and scattering (M3) modes.Three optical diffusers D1, D2 and D3 were placed at a common point of the optical axes of the LEDs in the plastic holders.
The LED mounts were then placed into the three holders.The holder for M1 mode was placed at the original light position, the second was fixed at the bottom to serve as illumination light source for M2 mode while the third was placed at the back, below the microscope handle, as light source for M3 mode.Resistors, transistors, operational amplifiers and diodes were put together on the stripboard to form an electronic circuit board (ECB) which controls the LEDs.This was placed beneath the microscope and linked to the various plastic holders with connectors.The NI-DAQ was also placed beneath the microscope with the input and output channels connected to the ECB.A binocular head which contains the eyepiece lenses was removed and a piece of black tape used in its place to prevent stray light.One end of the FB was placed at the emanating point of the transmission light source while the other end was linked to the holder for M3 mode.The trinocular head of the microscope was removed and replaced with the camera which was mounted on a support.The signals from the imager are controlled via the laptop computer using a developed Matlab program (R2014a Matlab 8.3.0.532,Mathworks Inc.).The user interface of this program is shown in Figure 2.

System Testing
Figure 3 shows spectral sensitivity range of the Guppy F-503B CMOS imager.The figure shows the 13 wavelengths range of the LEDs in the MSLEDIM.The highest spectral sensitivity peak is located at 590 nm.This renders the imager more sensitive to light at this wavelength, resulting in images of samples captured (at this wavelength) to be brighter than those captured at the other wavelengths.

Conclusions
Light sources used in MSI microscopy have restricted number of spectral bands associated with comparatively long acquisition times.This leads to sample photo-bleaching and photo-kinetics and ultimately unstable spectra.A retrofitted metallurgical microscope has been demonstrated for multi-spectral imaging.This new microscopic imaging system uses a relatively inexpensive set of 9 LEDs each as illuminating sources in reflection, transmission and scattering modes respectively.Each set of LEDs emits 13 wavelengths as light sources and is capable of acquiring megapixels microscopic images ranging from UV to NIR, making the imaging system an attractive tool for research.Images from this microscope are in grayscale colour model, which is device-independent and therefore colour information is unaffected by intensity manipulation.The results from the demonstrations show that the system is versatile and can potentially be used in biological and biomedical research.

Figure 1 .
Figure 1.Schematic diagram of the retrofitted metallurgical microscope using light emitting diodes for multi-spectral imaging.Three optical geometries M1, M2 and M3 provide multispectral illumination from the UV to NIR regions in reflection, transmission, and scattering modes respectively.The light from the sample mounted on the sample stage, SS, is collected through a reflective objective OB2.Detection is generally performed by a monochromatic CMOS imager, IMG

Figure 3 .
Figure 3. Spectral sensitivity range of the Guppy F-503B CMOS camera

Figure 4 .
Figure 4. Grayscale images acquired using the retrofitted metallurgical multispectral light-emitting diodes imaging microscope with 590 nm illumination source in (a) transmission, (b) scattering and (c) reflection modes.The dark spots in the images represent Plasmodium falciparum infected RBCs

Figure 6 .
Figure 6.(a) Red (R), (b) Green (G) and Blue (B) RGB-pixel histogram of intensities extracted from an image of a blood smear slide containing Plasmodium falciparum parasites in infected RBCs (iRBCs).The image was acquired using the retrofitted metallurgical microscope