FIELD

This invention relates to a colorimeter and a method of measuring colour of a sample in a container having an aperture.

BACKGROUND

When a sample is located in a relatively deep well, the sample is provided in a container where the numerical aperture of observable area of the sample is low (e.g. 1 :5). There are situations where reactions are performed on a substrate, such as a fdter, located in a deep well, where precursors or products change colour and it is desirable to monitor the process by optical means, such as by measuring the colour of the sample as the reaction progresses. However, currently available colorimeters are unable to measure the colour of such samples due to the narrow aperture of the container in which the sample is deeply set. This is because in such situations, illumination of the sample and observed light from the sample must both pass through the narrow aperture, and a single focusing system is unable to focus light from spatially separated light sources of different coloured lights onto the sample through the narrow aperture. There is thus a need for a colorimeter that is able to measure colour of a sample located in a container having a narrow aperture.

SUMMARY

According to a first aspect, there is provided a colorimeter to measure colour of a sample in a container having an aperture, the colorimeter comprising a receptacle to hold therein the sample in the container; a plurality of spatially separated light emitting diodes (LEDs) each configured to emit light of a different colour; a photodetector; a semi-transparent mirror positioned to reflect unfocused light from each of the plurality of LEDs through the aperture onto the sample and to allow passage of the scattered light from the sample through the semi transparent mirror to the photodetector, wherein the unfocused light reflected by the semi transparent mirror onto the sample and the scattered light from the sample to the photodetector pass through the aperture in opposite directions; a lens provided between the semi-transparent mirror and the photodetector to focus the scattered light from the sample onto the photodetector, wherein no light from the plurality of LEDs passes through the lens; a controller to control switching of light emission from each of the plurality of LEDs; and a digitizer to digitize output from the photodetector and transmit the digitized output to a computing device.

The numerical aperture of observation of the sample may be at least 1 :5.

The colorimeter may comprise a synchronous amplifier to amplify output from the photodetector in synchronization with light emission from the plurality of LEDs.

The colorimeter may comprise a software module provided in the controller to synchronize digitizing of output from the photodetector with switching of light emission from each of the plurality of LEDs by the controller.

The software module, the controller and the digitizer may be provided on an Arduino platform.

The controller, the digitizer and the synchronous amplifier may be provided on an Arduino platform

The colorimeter may be configured to be powered by a 5 Volt power source.

According to a second aspect, there is provided a method of measuring colour of a sample in a container having an aperture, the method comprising the steps of:

(a) placing the sample in the container in a receptacle;

(b) sequentially emitting unfocused light onto a semi-transparent mirror from each of a plurality of spatially separated light emitting diodes (LEDs) each configured to emit light of a different colour;

(c) controlling step (b) via a controller;

(d) reflecting the unfocused light through the aperture onto the sample via the semi transparent mirror;

(e) passing scattered light from the sample through the aperture and the semi-transparent mirror, wherein the unfocused light reflected by the semi-transparent mirror onto the sample and the scattered light from the sample pass through the aperture in opposite directions

(f) focusing the scattered light that has passed through the semi-transparent mirror onto a photodetector via a lens, wherein no light from the plurality of LEDs passes through the lens;

(g) digitizing output from the photodetector via a digitizer; and

(h) transmitting the digitized output to a computing device.

The method may comprise amplifying output from the photodetector via a synchronous amplifier in synchronization with step (a) prior to digitizing output from the synchronous amplifier by the digitizer.

The method may comprise providing the synchronous amplifier, the controller and the digitizer on an Arduino platform.

The method may comprise a software module synchronizing step (g) with step (b).

The method may comprise providing the software module, the controller and the digitizer on an Arduino platform.

For both aspects, the plurality of LEDs may be exchangeable.

The plurality of LEDs may comprise a first LED configured to emit light having a wavelength of 512nm, a second LED configured to emit light having a wavelength of 640nm, and a third LED configured to emit light having a wavelength of 850nm.

BRIEF DESCRIPTION OF FIGURES

In order that the invention may be fully understood and readily put into practical effect, there shall now be described by way of non-limitative example only exemplary embodiments of the present invention, the description being with reference to the accompanying illustrative drawings.

FIG. l is a schematic illustration of a first exemplary embodiment of a colorimeter.

FIG. 2 is a schematic illustration of a second exemplary embodiment of a colorimeter.

FIG. 3 is a photograph of samples of metallic nanostain on filters with different concentrations of bacteria.

FIG. 4 is a graph of colorimeter measurement of darkness of metallic nanostain on filters with different concentrations of bacteria.

FIG. 5 is a zoomed in section of higher intensity values of the graph of FIG. 4.

FIG. 6 is a flowchart of an exemplary method of measuring colour of a sample in a container having an aperture.

DETAILED DESCRIPTION

Exemplary embodiments of a colorimeter 100 and method 200 of measuring colour of a sample 10 in a container 12 having an aperture 16 will be described below with reference to FIGS. 1 to 6, where the same reference numerals refer to the same or similar parts.

As shown in FIGS. 1 and 2, the colorimeter 100 comprises a receptacle 20 to hold therein the sample 10 in its container 12. In certain situations, the container 12 has an aperture 16 that may be narrow (for example 4 mm in diameter or 2 mm in radius), while the sample 10 may be set rather deeply within the container 12 (for example 10 mm away from the aperture 16). The numerical aperture of observation or viewing angle is defined as the ratio of radius of the aperture 16 to the distance between the aperture 16 and the sample 10 in the container 12. In such situations, as a result of the low numerical aperture of observation of the sample 10 within the container 12, it is difficult to focus light through a focusing system onto the sample 10 from each of a plurality of spatially separated light emitting diodes (LEDs) 30 that are each configured to emit light of a different colour. Thus, in the presently disclosed colorimeter 100, the sample 10 is not illuminated through a focusing system. Instead, a semi transparent mirror 40 is provided to reflect unfocused light 39 from each of the spatially separated plurality of LEDs 30 through the aperture 16 onto the sample 10. An advantage of coloured illumination of the sample 12 by unfocused light 39 is that the unfocused light 39 is more tolerant of alignment errors such that misalignment of the sample 10 in its container 12 in the receptacle 20 does not change light intensity on the sample 10. A further advantage lies in the simplicity and lower cost of using a semi-transparent mirror to direct light onto the sample 10 as opposed to using a focusing system that requires use of one or more lenses

In an exemplary embodiment, the plurality of LEDs 30 may comprise a first LED configured to emit light having a wavelength of 512nm, a second LED configured to emit light having a wavelength of 640nm, and a third LED configured to emit light having a wavelength of 850nm. The plurality of LEDs 30 is preferably exchangeable so that desired colours of light can be used for desired purposes.

Emission of the unfocused light 39 is controlled by a controller 52 that controls switching of light emission from each of the plurality of LEDs 30. In an exemplary embodiment, sequential switching of the plurality of LEDs 30 may be controlled at a frequency of 22 Hz.

Notably, in the presently disclosed colorimeter 100, unfocused light 39 from the plurality of LEDs 30 does not pass through the sample 10 to another side of the sample 10 for detection, but is instead absorbed and re-emitted by the sample 10 as scattered light 19 in a direction opposite to the direction of incidence of the unfocused light 39 on the sample 10. The scattered light 19 from the sample 10 passes through the aperture 16 and also passes through the semi-transparent mirror 40 to be focused by a lens 70 onto a photodetector 60 comprised in the colorimeter 100. The semi-transparent mirror 40 is thus positioned to reflect unfocused light 39 from each of the plurality of LEDs 30 through the aperture 16 onto the sample 10 and allow passage of the scattered light 19 from the sample 10 through the semi-transparent mirror 40 and the lens 70 onto the photodetector 60. Notably, the unfocused light 39 reflected by the semi-transparent mirror 40 onto the sample 10 and the scattered light 19 from the sample 10 both pass through the aperture 16 of the container 12 in opposite directions, as can be seen in FIGS. 1 and 2.

In the colorimeter 100, the lens 70 is provided between the semi-transparent mirror 40 and the photodetector 60 to focus the scattered light 19 from the sample 10 onto the photodetector 60. Notably, no light from the plurality of LEDs 30 passes through the lens 70 as all light provided by the plurality of LEDs 30 and incident on the sample 10 is unfocused light 39. The receptacle 20, semi-transparent mirror 40, lens 70 and photodetector 60 may be provided in an optical box 90 in the colorimeter 100. In an exemplary embodiment, numerical aperture of the optical system formed by the components in the optical box 90 may be as low as 1 :5 when the aperture 16 has a radius of 2 mm and the sample 10 is 10 mm away from the aperture 16. It should be noted that the colorimeter 100 and method 200 will work with other samples 10 having numerical apertures of observation of at least 1 :5, including ratios such as 1 :4, 1 :3 and so on. In a first exemplary embodiment, as shown in FIG. 1, the colorimeter 100 further comprises a synchronous amplifier 80 that amplifies output signals from the photodetector 60 in synchronization with light emission from the plurality of LEDs 30. The synchronous amplifier 80 may give an output voltage in each channel ranging from 0 to 2 Volts that is proportional to intensity of scattered light 19 from the sample 10. In this embodiment, a digitizer 54 is provided to digitize the output voltage from the amplifier 80 and transmit the digitized output to a computing device (not shown). The digitizer 54 and the synchronous amplifier 80 may be provided on the Arduino platform 50 together with the controller 52.

In a second exemplary embodiment, as shown in FIG. 2, the colorimeter 100 may comprise a digitizer 54 provided to digitize output from the photodetector 60 and transmit the digitized output to a computing device, as well as a software module (not shown) provided in the controller 52 and configured to synchronize digitizing of output from the photodetector 60 by the digitizer 54 (i.e. synchronous digitizing) with switching of light emission from each of the plurality of LEDs 30 by the controller 52. The software module, the digitizer 54 and the controller 52 may be provided on an Arduino platform 50. Compared to using a synchronous amplifier followed by digitizing the amplifier output, synchronous digitizing simplifies the electronic circuitry and reduces cost of the colorimeter 100. An example of an Arduino software programme code for controlling individual colour illumination from the plurality of LEDs and digitizing output from the photodetector 60 is given below: long inputl = 0;

long input2 = 0 ;

long input3 = 0;

long input4 = 0;

long input5 = 0;

long ledread[7] =

1 0 , 0 , 0 , 0 , 0 , 0 , 0 ) ;

int reads[7] = 10,0,0,0,0,0,0)

int i = 0 ;

Int takes = 0;

int inputPinl = A0;

Int lnputPin2 = A1 ;

int inputPin3 = A2 ;

int inputPin4 = A3 ;

int inputPin5 = A4 ; int lednow = 2 ;

int ledperiod = 20;

int ledcount = 0;

int inputmode = LOW;

float vail;

float val2;

float val3;

float val4;

float dark;

int inByte = 0 ;

void setup ( ) {

// put your setup code here, to run once:

Serial . begin ( 9600 ) ;

pinMode (inputPinl, INPUT); pinMode ( inputPin2 , INPUT); pinMode (inputPin3, INPUT); pinMode ( inputPin4 , INPUT); pinMode ( inputPin5 , INPUT); pinMode (2, OUTPUT);

pinMode ( 3 , OUTPUT);

pinMode ( 4 , OUTPUT);

pinMode ( 5 , OUTPUT);

pinMode ( 6, OUTPUT);

pinMode (7, INPUT);

digitalWrite (lednow, HIGH);

}

void loop ( ) {

inputl = inputl +

analogRead ( inputPinl ) ;

input2 = input2 +

analogRead ( inputPin2 ) ;

input3 = input3 +

analogRead ( inputPin3 ) ;

input4 = input4 +

analogRead ( inputPin4 ) ;

takes = takes + 1;

if (ledcount > 2)

{ ledread [lednow] =

ledread [ lednow] +

analogRead ( inputPin5 ) ; reads [lednow] = reads [lednow]

+i; }

ledcount = ledcount +1;

if (ledcount > ledperiod)

{

digitalWrite (lednow, LOW); lednow = lednow + 1;

If (lednow > 6) lednow = 2; digitalWrite ( lednow, HIGH); ledcount = 0;

}

if ( Serial . available ( ) > 0)

{

inByte = Serial . read () ;

inputmode = digitalRead ( 7 ) ; if (inputmode == HIGH)

{dark = (float) ledread[6] / reads [ 6 ] ;

vail = (float) dark - ledread[2] / reads [2];

val2 = (float) dark - ledread[3] / reads [3];

val3 = (float) dark - ledread[4] / reads [4];

val4 = (float) dark - ledread[5] / reads [5];) else

{vail = (float) Inputl/takes ; val2 = (float) input2/takes ; val3 = (float) input3/takes ; val4 = (float)

input4/takes ; }

Serial . print (val 1 ) ;

Serial. printC',") ;

Serial . print (val2 ) ;

Serial. printf',") ;

Serial . print (val3 ) ;

Serial. printf',") ;

Serial .printIn (val4 ) ;

inputl = 0;

input2 = 0; input3 = 0 ;

input4 = 0 ;

takes = 0;

for (int i=2; i<=7 ; i++)

{ ledread [ i ] =0 ; reads[i]=0;)

}

if (takes > 11000)

{

inputl = 0;

input2 = 0;

input3 = 0 ;

input4 = 0 ;

takes = 0;

for (int i=2 ; i<=6; i++)

{ledread[i] =0; reads[i]=0;)

}

}

In all embodiments, the colorimeter 100 provides digital colour synchronization of illumination and detection of the sample 10. The whole colorimeter 100 can be powered using a single 5 V source (e.g. from a USB port or battery) and has a low power consumption of 300 mW. The colorimeter 100 is simple and compact, and in an exemplary embodiment, may weigh about 400 grams and have a compact size of 20 cm x 12 cm x 7 cm. The colorimeter 100 may also be modified to function as a fluorimeter by proper choice of LEDs and placing an appropriate optical filter before the photodetector 60.

In an example of use, the colorimeter 100 was applied for quantitative bacteria detection. Specifically, the colorimeter 100 was used to analyse darkness of metallic nanostains on filter membranes, wherein each membrane was located at 10 mm deep in a filter having an aperture 16 with a diameter of 4mm. The filter membrane samples 10 held bacteria at different concentrations. Recording of signals using the colorimeter 100 began without a sample. As outlined in FIG. 6, the filter samples 10 were then inserted or placed, one by one, into the receptacle 20 of the optical box 90 (201) and held there for 60 seconds each to perform the illumination and detection of each sample 10. The illumination and detection process comprised sequentially emitting unfocused light 39 onto the semi-transparent mirror 40 from each of the plurality of spatially separated LEDs 30 (202) under the control of the controller 52 (203), and reflecting the unfocused light 39 through the narrow aperture 16 onto the sample 10 via the semi-transparent mirror 40 (204). Scattered light 19 from the sample 10 was then passed through the aperture 16 and the semi-transparent mirror 40, wherein the unfocused light 39 reflected by the semi-transparent mirror 16 onto the sample 10 and the scattered light 19 from the sample 10 pass through the narrow aperture 16 in opposite directions (205), and the scattered light 19 from the sample 10 was focused onto the photodetector 60 via the lens 70, wherein no light from the plurality of LEDs 30 passes through the lens 70 (206). Output from the photodetector 60 was digitized via the digitizer 54 (207) and transmitted to a computing device (208). It was observed that when a filter sample 10 was inserted into the colorimeter 100, the intensity of scattered light 19 was seen to increase. Darker filter membrane samples 10, such as those having 10 ⁶ and 10 ⁷ bacteria concentration levels, produced lower increase of scattered light 19. Light intensity obtained from the filter samples 10 was normalised against light intensity obtained with a blank filter having 0 bacteria concentration level, and the normalised intensity values are shown in the graph of FIG. 4. The graph in FIG. 5 shows a zoomed in view of the higher normalized intensity values of the graph in FIG. 4, i.e., from 700 and above. From FIG. 5, it can be seen that the noise level of the colorimeter 100 is around 1% of the blank value having a bacteria concentration level of 0.

Notably, the present colorimeter 100 relies on using unfocused light 39 and a semi transparent mirror 40 to direct unfocused light 39 onto the sample 10. This is despite unfocused light 39 and a semi-transparent mirror 40 typically being not the illumination components of choice in optical systems due to the low light intensity provided by unfocused light that is worsened by a drop in light intensity when light is reflected off the semi transparent mirror, resulting in sample measurement being made more difficult. Nevertheless, the present colorimeter 100 is able to use unfocused light 39 and a semi transparent mirror 40 to illuminate the sample 10, thus overcoming the difficulty of focusing spatially separated colour light sources on a deeply set sample in a container having a narrow aperture, while also providing a simple, cost-effective and compact device to obtain colour measurements of samples 10 without needing a complex illumination system.

Whilst there has been described in the foregoing description exemplary embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations and combination in details of design, construction and/or operation may be made without departing from the present invention. For example, while the above disclosed colorimeter and method are particularly able to measure colour of samples that have a small angle of observation, such as deep set samples in containers having narrow apertures, the colorimeter and method may also be used to measure colour of samples of other configurations without being limited to samples in containers having narrow apertures.